IMAGING-PAM M-series Chlorophyll Fluorometer - Walz

IMAGING-PAM M-series Chlorophyll Fluorometer - Walz
IMAGING-PAM
M-series
Chlorophyll Fluorometer
Instrument Description
and
Information for Users
2.152 / 07.06
5. revised Edition: March 2014
imag-m-series0e_3.doc
 Heinz Walz GmbH, 2014
Heinz Walz GmbH  Eichenring 6  91090 Effeltrich  Germany
Phone +49-(0)9133/7765-0  Telefax +49-(0)9133/5395
E-mail [email protected]  Internet www.walz.com
Printed in Germany
CONTENTS
1 Safety instructions ........................................................................ 1 1.1 General safety instructions ....................................................... 1 1.2 Special safety instructions ........................................................ 2 2 Introduction .................................................................................. 3 3 Components of the IMAGING-PAM MAXI-version .................. 7 3.1 Control Unit IMAG-CG ........................................................... 9 3.2 LED-Array Illumination Unit IMAG-MAX/L and IMAGMAX/LR ................................................................................ 11 3.3 CCD Camera IMAG-K6 and objective K6-MAX.................. 15 3.4 CCD Camera IMAG-K7 and objectives K7-MAX/Z and K7MAX/S ................................................................................... 17 3.5 Mounting Stand with Eye Protection IMAG-MAX/GS ......... 19 3.6 Leaf Distance Holder IMAG-MAX/B ................................... 24 3.7 Notebook PC IMAG-PC ........................................................ 25 3.8 Adapter IMAG-MAX/GWK .................................................. 26 4 Components of the IMAGING-PAM MINI-version .................. 27 4.1 Multi Control Unit IMAG-CG ............................................... 27 4.2 MINI-Head LED-Array IMAG-MIN/B and IMAG-MIN/R .. 28 4.3 CCD Camera IMAG-K6 or IMAG-K7 .................................. 30 4.4 IMAG-MIN/GFP with IMAG-K6 .......................................... 32 4.5 Leaf Holder IMAG-MIN/BK with Grip Holder..................... 38 4.6 Adapter for GFS-3000 (IMAG-MIN/GFS) ............................ 42 4.7 ImagingWin software versions for various types of MINIVersion ................................................................................... 44 5 Components of the IMAGING-PAM MICROSCOPY-version . 46 5.1 Multi Control Unit IMAG-CG ............................................... 47 5.2 CCD Camera IMAG-K6 ........................................................ 47 5.3 Axio ScopeA.1 Epifluorescence Microscope ......................... 49 5.3.1 Reflector Modules .............................................................. 51 I
CONTENTS
5.3.2 Assembling of beam splitter and filters.............................. 52 5.3.3 Mounting of the reflector module ...................................... 54 5.4 LED Modules ......................................................................... 56 5.4.1 Adjustment of brightness by grey filters ............................ 56 5.4.2 Integration of LED modules into Axio Scope.A1 .............. 58 5.4.3 Connecting LED modules with IMAG-CG ....................... 60 5.4.4 Switching LED modules for measurements ....................... 60 5.4.5 IMAG-RGB ....................................................................... 60 6 How to get started ...................................................................... 63 6.1 Connecting the cables ............................................................ 63 6.2 Software installation............................................................... 64 6.2.1 Installation and Starting of ImagingWin ............................ 64 6.2.2 Installation of camera driver .............................................. 66 6.3 First steps and examples of routine measurements ................ 66 7 ImagingWin ................................................................................ 81 8 IMAGINGWIN - System Operation .......................................... 83 8.1 Definition of New Record ...................................................... 83 8.1.1 Fo, Fm ................................................................................ 83 8.1.2 New Record........................................................................ 84 8.1.3 Measure .............................................................................. 84 8.2 Functions applying to the View-mode ................................... 85 8.3 Light controls ......................................................................... 87 9 IMAGINGWIN - Register Cards ............................................... 90 9.1 Image-window........................................................................ 90 9.1.1 Different types of images ................................................... 90 9.1.1.1 Current fluorescence yield, Ft ........................................ 91 9.1.1.2 Dark fluorescence yield, Fo ........................................... 91 9.1.1.3 Fluorescence yield, F ..................................................... 92 II
CONTENTS
9.1.1.4 Maximal fluorescence yield, Fm .................................... 92 9.1.1.5 Maximum fluorescence yield, Fm' ................................. 93 9.1.1.6 Maximal PS II quantum yield, Fv/Fm ........................... 93 9.1.1.7 Effective PS II quantum yield, Y(II) .............................. 94 9.1.1.8 Quantum yield of regulated energy dissipation, Y(NPQ)95 9.1.1.9 Quantum yield of nonregulated energy dissipation,
Y(NO) ............................................................................ 96 9.1.1.10 Absorptivity, Abs. .......................................................... 97 9.1.1.11 Apparent rate of photosynthesis, PS/50 ......................... 99 9.1.1.12 NIR light remission, NIR ............................................. 100 9.1.1.13 Nonphotochemical quenching, NPQ/4 ........................ 101 9.1.1.14 Red light remission, R.................................................. 102 9.1.1.15 Coefficient of nonphotochemical quenching, qN ........ 103 9.1.1.16 Coefficient of photochemical quenching, qP ............... 104 9.1.1.17 Coefficient of photochemical quenching, qL ............... 105 9.1.1.18 Inhibition, Inh. ............................................................. 106 9.1.2 Image capture and analysis .............................................. 107 9.1.2.1 Measure Abs. ............................................................... 107 9.1.2.2 Area of Interest, AOI ................................................... 108 9.1.2.3 Select: Fluorescence or Live Video ............................. 110 9.1.2.4 Zoom ............................................................................ 112 9.1.2.5 Cursor........................................................................... 113 9.1.2.6 Analysis ....................................................................... 113 9.2 Kinetics window................................................................... 116 9.3 Light Curve window ............................................................ 122 9.4 Report window ..................................................................... 128 9.5 Settings window ................................................................... 132 9.5.1 Light parameters .............................................................. 133 III
CONTENTS
9.5.2 Gain and Damping ........................................................... 136 9.5.3 Absorptivity ..................................................................... 137 9.5.4 Slow Induction parameters ............................................... 138 9.5.5 Image Correction.............................................................. 138 9.5.6 Image Transformation ...................................................... 141 9.5.7 Battery .............................................................................. 141 9.5.8 Display parameters ........................................................... 142 9.5.9 Go Speed .......................................................................... 144 9.5.10 PS Limit ........................................................................... 144 9.5.11 Inh. Ref. AOI ................................................................... 145 9.5.12 Yield Filter ....................................................................... 145 9.5.13 Fm Factor ......................................................................... 146 9.5.14 F Factor ............................................................................ 149 9.5.15 Reset Default Settings, Open or Save User Settings ........ 152 9.6 High Sens. window .............................................................. 153 9.6.1 Special SP-Routine .......................................................... 154 9.6.2 Fo Averaging.................................................................... 156 9.6.3 Fv/Fm Contrast Enhancement by Background Suppression156 9.7 RGB-Fit window .................................................................. 157 9.7.1 RGB Gain ......................................................................... 160 9.7.2 Fit Correction ................................................................... 160 10 IMAGINGWIN - Menu Bar ..................................................... 163 10.1 File ....................................................................................... 163 10.1.1 Transfer FoFm.................................................................. 163 10.1.2 Using Skript files - Load Script/Run Script ..................... 163 10.1.3 Exit ................................................................................... 171 10.2 Options ................................................................................. 172 10.2.1 Ruler ................................................................................. 172 IV
CONTENTS
10.2.2 Scale ................................................................................. 172 10.2.3 Info Icons ......................................................................... 173 10.2.4 Mean over AOI ................................................................ 173 10.2.5 Define AOI-array geometry ............................................. 175 10.2.6 Create AOI array: ............................................................. 176 10.3 Al-List .................................................................................. 178 10.3.1 LED currents / PAR values .............................................. 178 10.3.2 Light Calibration .............................................................. 180 10.4 Recalc ................................................................................... 181 10.5 Transect ................................................................................ 182 11 List of key commands .............................................................. 185 12 Technical specifications ........................................................... 186 12.1 Components used in all Versions ......................................... 186 12.1.1 Control Unit IMAG-CG ................................................... 186 12.1.2 IMAG-K7 ......................................................................... 187 12.1.3 IMAG-K6 ......................................................................... 187 12.1.4 Windows Software ImagingWin ...................................... 187 12.1.5 Battery Charger 2120-N ................................................... 188 12.2 Components specifically relating to Maxi-version .............. 189 12.2.1 LED-Array Illumination Unit IMAG-MAX/L ................. 189 12.2.2 LED-Array Illumination Unit IMAG-MAX/LR .............. 190 12.2.3 Optional filter plate IMAG-MAX/F (only for IMAGMAX/L!) .......................................................................... 191 12.2.4 External 300 W Power Supply ......................................... 191 12.2.5 K7-MAX/Z....................................................................... 191 12.2.6 K7-MAX/S ....................................................................... 192 12.2.7 K6-MAX .......................................................................... 192 12.2.8 K6-MAX/M and K7-MAX/M.......................................... 193 V
CONTENTS
12.2.9 Mounting Stand with Eye Protection IMAG-MAX/GS... 193 12.2.10 IMAG-MAX/B.............................................................. 194 12.2.11 ST-101........................................................................... 194 12.2.12 Transport Box IMAG-MAX/T...................................... 194 12.2.13 IMAG-MAX/GWK1 ..................................................... 195 12.3 Components specifically relating to MINI-version .............. 195 12.3.1 IMAG-MIN/B .................................................................. 195 12.3.2 IMAG-MIN/R .................................................................. 196 12.3.3 IMAG-MIN/GFP ............................................................. 196 12.3.4 K7-MIN............................................................................ 197 12.3.5 K6-MIN............................................................................ 197 12.3.6 K6-MIN/FS ...................................................................... 197 12.3.7 K7-MIN/M and K6-MIN/M............................................. 198 12.3.8 IMAG-S ........................................................................... 198 12.3.9 IMAG-MIN/ST ................................................................ 198 12.3.10 ST-1010......................................................................... 199 12.3.11 IMAG-MIN/BK ............................................................ 199 12.3.12 IMAG-MIN/GFS .......................................................... 199 12.4 Components specifically relating to MICROSCOPY-versions199 12.4.1 IMAG-AXIOSCOPE ....................................................... 199 12.4.2 IMAG-L470M .................................................................. 200 12.4.3 IMAG-L625M .................................................................. 200 12.4.4 IMAG-RGB ..................................................................... 200 12.4.5 IMAG-AX-REF ............................................................... 201 13 Warranty ................................................................................... 202 13.1 Conditions ............................................................................ 202 13.2 Instructions to obtain Warranty Service, .............................. 203 VI
CHAPTER 1
SAFETY INSTRUCTIONS
1 Safety instructions
1.1
General safety instructions
1.
Read the safety instructions and the operating instructions first.
2.
Pay attention to all the safety warnings.
3.
Keep the device away from water or high moisture areas.
4.
Keep the device away from dust, sand and dirt.
5.
Always ensure there is sufficient ventilation.
6.
Do not put the device anywhere near sources of heat.
7.
Connect the device only to the power source indicated in the
operating instructions or on the device.
8.
Clean the device only according to the manufacturer’s
recommendations.
9.
If the device is not in use, remove the mains plug from the
socket.
10. Ensure that no liquids or other foreign bodies can find their way
inside the device.
11. The device should only be repaired by qualified personnel.
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CHAPTER 1
1.2
SAFETY INSTRUCTIONS
Special safety instructions
The IMAGING-PAM is a highly sensitive research instrument
which should be used only for research purposes, as specified in this
manual. Please follow the instructions of this manual in order to
avoid potential harm to the user and damage to the instrument.
Never use the Multi Control Unit IMAG-CG with more than one
Measuring Head plugged in at the same time.
The IMAGING-PAM employs strong blue light for excitation of
chlorophyll fluorescence, for driving photosynthetic electron
transport and for transient saturation of photosynthetic energy
conversion (Saturation Pulse method). In order to avoid harm to your
eyes, please avoid looking directly into this light, particularly during
Saturation Pulses.
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INTRODUCTION
2 Introduction
The IMAGING-PAM Chlorophyll Fluorometer is specialized for
the study of two-dimensional heterogeneities of photosynthetic
activity. The Imaging-PAM M-series covers a wide range of
applications. Large scale samples with areas exceeding multiwell
plate format can be imaged as well as microscopically small samples
at the level of single cells. MAXI-, MINI- and MICROSCOPYversions have been issued that are operated with the same Multi
Control Unit IMAG-CG. Like all PAM fluorometers, the
Imaging-PAM applies pulse-amplitude-modulated measuring light
for assessment of chlorophyll fluorescence yield. The same LEDs not
only serves for generation of the pulse-modulated measuring light,
but also for actinic illumination driving photosynthesis and for
Saturation Pulses transiently saturating energy conversion at
Photosystem II (PS II) reaction centers. The Saturation Pulse method
provides a non-destructive means of analyzing the photosynthetic
performance of plants. It allows to assess the quantum yield of
energy conversion at PS II reaction centers, which is affected by
numerous intrinsic and environmental parameters, like the
physiological health, light conditions and various stress factors.
Since the introduction of PAM fluorometry in 1985, a large amount
of literature has been published on the practical use of this method in
many fields of plant science. In principle, with all IMAGING-PAM
Fluorometers the same kind of measurements are possible as with
Standard-PAM Fluorometers (e.g. Dual-PAM, PAM-2500 or
MINI-PAMII), most users already may be accustomed to. Hence,
also with all versions of the IMAGING-PAM M-series the
characteristic fluorescence levels Fo, Fm and Fm' can be assessed
and quenching coefficients derived. Also the PS II quantum yield
Fv/Fm (or F/Fm') can be determined and Induction Curves as well
as Light Saturation Curves with quenching analysis can be measured.
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INTRODUCTION
The most essential new information provided by chlorophyll
fluorescence imaging relates to the detection of lateral
heterogeneities of fluorescence parameters which reflect
physiological heterogeneities. It has been known for some time, that
even physiologically healthy leaves are "patchy" with respect to
stomatal opening. Furthermore, stress induced limitations, which
eventually will lead to damage, are not evenly distributed over the
whole leaf area. Fluorescence imaging may serve as a convenient
tool for early detection of such stress induced damage. Hence,
favorite fields of application of fluorescence imaging are plant stress
physiology and plant pathology. An outstanding feature of the
Imaging-PAM distinguishing it from standard PAM fluorometers is
the possibility of parallel assessment of several samples under
identical conditions. For this applications the MAXI-version is
particularly well suited, e.g. for the screening of mutants in plant
molecular biology and for the assessment of samples in multi-well
plates, e.g. 96-well plates in ecotoxicological studies. The MINIversion is particularly compact and easy to handle and therefore best
suited for field applications. MINI- and MAXI-versions also can be
readily adapted for simultaneous measurements with the GFS-3000
Gas Exchange Measuring System. The MICROSCOPY-versions
provide the opportunity of imaging heterogeneities at the level of
single cells (e.g. guard-cells or algae cells). Using the RGB-Head it
is even possible to differentiate between different pigment types, like
diatoms, chlorophytes and cyanobacteria.
The MAXI- and MINI-versions not only measure fluorescence,
but also provides an estimate of incident light absorptivity. This
aspect is particularly important when dealing with lateral
heterogeneities of chlorophyll content often accompanying stress- or
pathogen-induced damage. For assessment of absorptivity of the
incident photosynthetically active radiation (PAR), the same sample
is irradiated with diffuse near-infrared (NIR) and red light, the
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INTRODUCTION
remitted parts of which are imaged with the same CCD-camera
which serves for fluorescence imaging.
As the different versions of the Imaging-PAM M-series are
optimized for largely different sample sizes, they apply quite
different LED sources. While the same methodology applies for all
versions, there are very different power requirements for appropriate
intensities of measuring, actinic and saturation pulse light. The
MAXI-IMAGING-PAM is equipped with an extremely powerful
array of 44 high power (3 W) Luxeon LEDs, each of which is driven
with currents up to 1.6 A. The same type of LEDs is also used in the
MINI-IMAGING-PAM (featuring 12 LEDs illuminating 24 x 32 mm
area) and various versions of the MICROSCOPY-IMAGING-PAM
(with a single LED coupled in various ways to the excitation port of
various types of Epifluorescence Microscopes). Therefore, the same
Power-and-Control Unit (IMAG-CG) can be used for these different
measuring heads.. The new IMAG-CG also features an output for
controlling a special RGB-LED-Lamp that is equipped with its own
LED drivers.
As the various versions of the Imaging-PAM M-series put
different demands on the sensitivity, different CCD-cameras are
employed. Two different CCD-cameras are available for the MAXIand MINI-version. For high sensitivity applications (e.g.
phytotoxicity bioassay using multiwell plate with algae suspensions)
a 2/3" CCD camera (1392x1040 pixel with 4-pixel-binning) is
recommended, which is also generally used with the
MICROSCOPY-versions. For standard applications an economical
1/2" CCD camera (640x480 pixel) is available, which is particularly
useful in conjunction with a powerful zoom objective.
This manual provides some essential information on the
components of the different versions of the Imaging-PAM M-series
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CHAPTER 2
INTRODUCTION
and on the ImagingWin software. While the latter in principle applies
to all versions, it also offers some special features that can be used
with particular versions only (e.g. RGB-Fit images using the
MICROSCOPY/RGB-version). As it is most frequently used and
also featuring the widest range of applications/configurations, there
is some emphasis on the MAXI-version in this manual.
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CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
3 Components of the IMAGING-PAM MAXIversion
In the following chapters the three different versions: the MAXI-,
MINI- and Microscopy-version of the Imaging-PAM M-series are
described. As the MAXI-version is most frequently used, those
components that are common to all versions are dealt with in this
chapter on the MAXI-version.
The basic measuring system of the MAXI-IMAGING-PAM consists
of:
1) Control Unit IMAG-CG with Battery Charger 2120-N and
External 300 W Power Supply
2) LED-Array Illumination Units IMAG-MAX/L (blue) or IMAGMAX/LR (red). A useful accessory for measurements with IMAGMAX/L investigating mirroring samples (like multiwell plates filled
with algae suspensions) is the Filter Plate IMAG-MAX/F, which
absorbs the small fraction of red light contained in the blue LED
light.
3) CCD Cameras IMAG-K7 or IMAG-K6 with accessories and
mounting sets
4) Mounting Stand with Eye Protection IMAG-MAX/GS,
laboratory stand ST-101 or Leaf Distance Holder IMAG-MAX/B
5) PC with ImagingWin-software
Combining imaging and gas exchange measurements, the adapter
IMAG-MAX/GWK for gas exchange chamber GWK1 is available.
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CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
Fig. 1: Transport Box IMAG MAX/T provided with each of the basic
Imaging systems offers enough space for all essential components
(except PC)
For transport of components 1 - 4, as well as of all essential
cables, the Transport Box IMAG-MAX/T is provided.
Note:
In the LED-Array Illumination Unit an
Adapter Ring may be inserted, which is
not screwed. To avoid damage to the
instrument, please do not take it out of the
box by using this hole as a handle.
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CHAPTER 3
3.1
COMPONENTS OF THE MAXI-VERSION
Control Unit IMAG-CG
The Control Unit IMAG-CG contains a rechargeable Li-ion
battery (14.4 V/6 Ah). The main printed circuit board of the IMAGCG contains a RISC processor microcontroller, the power supply for
the CCD camera and the LED drivers for the Maxi-LED-Arrays. The
same LED drivers also serve for the alternative MINI- or
MICROSCOPY-Heads (MINI- and RGB-sockets at the backside of
the instrument). The Control Unit provides the power for driving the
LED-Arrays of all members of the M-Series systems except the
MAXI-IMAGING-PAM, which is driven by an external 300 W
Power Supply.
Note:
Never switch on the Control Unit IMAG-CG with more
than one Measuring Head connected at the same time.
Fig. 2: Front and rear side views of the Control Unit IMAG-CG
The functional elements at the front side of the instrument are:
POWER
Power on/off switch; when switched on, the green
status LED at the right hand side of the switch
lights up.
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COMPONENTS OF THE MAXI-VERSION
CHARGE-LED
Charge LED lights up red while battery is charged
with the help of the Battery Charger 2120-N. When
the battery is fully charged, the LED lights up
green.
CHARGE
Socket for connecting Battery Charger 2120-N. An
external 12 V battery cannot recharge the internal
14.4 V Li-ion battery.
Note:
Please avoid charging the internal Li-ion battery while the
IMAGING-PAM is switched on.
MAXI-HEAD
Socket for connecting LED-Array Illumination Unit
IMAG-MAX/L (MAXI-Head).
CAMERA
Socket for camera cable via which trigger signals
and power is transferred to the CCD-camera of the
MAXI-Head or MICROSCOPY-IMAGING-PAM.
At the rear side of the housing, the Control Unit features three
sockets, which apply for use of alternative Measuring-Heads:
MINI-HEAD
Socket for connecting MINI- Heads as well as the
IMAG-L470M or IMAG-L625M LED lamps of
the MICROSCOPY-version
RGB-HEAD
Socket for connecting the optional Red/Green/
Blue-Head of the MICROS-COPY-version
Ext. out
Socket for connecting an optional external light
source
When using MINI-Head, the camera can directly be mounted on
the top of the Control Unit housing. For this purpose a wing-screw is
provided.
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CHAPTER 3
3.2
COMPONENTS OF THE MAXI-VERSION
LED-Array Illumination Unit IMAG-MAX/L and IMAGMAX/LR
Fig. 3: Front view of IMAG MAX/L with objective lens of CCD-camera
protruding through central opening
The LED-Array Illumination Unit IMAG-MAX/L features 44
high-power royal-blue (450 nm) LED-lamps equipped with
collimating optics, which are arranged for maximal intensity and
homogeneity at 17 - 20 cm distance to the object plane. These LEDlamps provide the pulse-modulated blue excitation light and at the
same time serve for actinic illumination and Saturation Pulses. In
addition, there are 4 groups of 8 LEDs providing the pulse
modulated light for assessment of PAR-Absorptivity (see 9.1.1.10).
These LEDs are arranged in pairs, with each pair featuring a red
(660 nm) and a near-infrared (780 nm) LED. The lenses of these
LEDs are removed in order to obtain homogenous illumination of the
sample. While only a relatively small amount of this light is remitted
from the sample to the CCD-camera, this is sufficient to give good
signals, as both wavelengths can pass the red long-pass filter in front
of the CCD-chip, in contrast to the blue excitation light.
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COMPONENTS OF THE MAXI-VERSION
A useful accessory for measurements with mirroring samples
(like multiwell plates filled with algae suspensions) is the Filter Plate
IMAG-MAX/F that absorbs the small fraction of red light contained
in the blue LED light. This plate can be mounted with 4 screws at the
front side of the Illumination Unit. Unavoidably, the effective PARvalues are lowered by about 15% by the filtering.
Fig. 4: Mounting of IMAG-MAX/F. Four screws are provided with the
filter plate
The LED-Array Illumination Unit IMAG-MAX/LR
has a
similar organization as IMAG-MAX/L but features 44 red (650 nm)
high-power LED-lamps and the four groups of red (660 nm) and a
near-infrared (780 nm) LEDs. The IMAG-MAX/LR includes the
filterplate IMAG-MAX/FR .
Both Illumination Units feature two cables, which connect to the
MAXI-HEAD socket at the front side of the Control Unit IMAG-CG
and to the external 300 W power supply.
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COMPONENTS OF THE MAXI-VERSION
Fig. 5: IMAG MAX/L in conjunction with IMAG MAX/GS and mounted
CCD camera IMAG MAX/K6
On the top side of the Illumination Units a fan is located that
serves for cooling the aluminum plate on which the slugs of the highpower LEDs are mounted. The adapter, on which the CCD-camera is
mounted, at the rear side features an adapter hole for installation of a
15 mm  metal bar, which may serve for mounting the Measuring
Head independently of the Mounting Stand with Eye Protection
(IMAG-MAX/GS) (see Fig. 6). This bar is fixed with a hex-nut
screw, for which a corresponding key is provided. Please note that
the nut should press against the flattened side of the bar.
Fig. 6: Measuring Head consisting of LED-Array Illumination Unit and
CCD-camera mounted via 15 mm Ø metal bar on optional stand
(Stand with Base Plate ST 101)
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COMPONENTS OF THE MAXI-VERSION
Warning:
When used without Mounting Stand and Eye protection,
the user should avoid looking directly into the LED-Array
Illumination unit.
The user is urgently advised to ensure an alternative eye
protection, which is effective in applications where the
standard IMAG-MAX/GS cannot be applied.
When mounted independently from the Mounting Stand with Eye
Protection (IMAG-MAX/GS, see 3.5), the working distance of the
LED-Array can be adjusted between 14.5 and 22.5 cm, resulting in
imaged areas between 7.5 x 10 and 11 x 15 cm. Optimal light field
homogeneity is obtained at the standard distance of 18.5 cm (imaged
area 9 x 12 cm), with +/- 7% maximal deviation of intensity from the
mean value.
A fixed standard distance of 18.5 cm is provided when the
IMAG-MAX/L is mounted on the IMAG-MAX/GS (see 3.5). As the
LED-intensity is defined by the ImagingWin software, the photon
flux density (PAR) at this standard distance is well defined. Small
variations in the PAR-distribution as well as the unavoidable
vignetting effect of the camera objective lens can be measured and
corrected for by software (see Image Correction in section 9.5.5).
Three different Image Corrections are supported by the ImagingWin
software for three different distances: Type 1, Type 2 and MAXI
(chapter 9.5.5).
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COMPONENTS OF THE MAXI-VERSION
Note:
In the LED-Array Illumination Unit
an Adapter Ring may be inserted,
which is not screwed. To avoid
damage to the instrument, please do
not pull the Imag-MAX/L it out of
the box by using this hole as a
handle.
3.3
CCD Camera IMAG-K6 and objective K6-MAX
Fig. 7: CCD Camera IMAG-K6
The CCD Camera IMAG-K6 (Allied Vision Technologies)
features a 2/3" chip with 1392 x 1040 pixel. The data are digitized
within the camera and transferred via ethernet interface (GigEVision®) to the PC. The sockets for connecting the GigE-cable as
well as the camera-control-cable (round-shaped connector) are
located at the rear side of the camera.
As the CCD-chip features 1392 x 1040 pixel in contrast to the
resulting 640 x 480 pixel of the ImagingWin fluorescence images,
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COMPONENTS OF THE MAXI-VERSION
the signals of 4 pixel are combined (2 x 2 pixel binning), thus
providing outstanding sensitivity. This feature is particularly useful
for reliable assessment of the "dark fluorescence" parameters Fo, Fm
and Fv/Fm.
Note:
The aperture of the IMAG-K6 standard lens can be closed. This
increases the depth of focus, but decreases the signal intensity. For
plane objects, the setting of the aperture should always completely
open.
The camera is equipped with a metal angle bar (black anodized),
which serves for mounting it on the corresponding adapter on the
IMAG-MAX/L Illumination Unit. Mounting the IMAG-K6 camera
for use in conjunction with MINI- or MICROSCOPY- Version are
described in the corresponding chapters. If the camera is shared
between different M-series instruments and setups, it might be
necessary to adapt camera objective and the distance ring between
objective and the CCD chip.
A Cosmicar-Pentax objective with 12.5 mm focal length (F=1.4)
is used in conjunction with the LED-Array Illumination Unit IMAGMAX/L or IMAG-MAX/LR
(Fig. 8). A 3 mm RG 665 (Schott)
color glass filter serves as long-pass filter for protecting the CCDchip from blue excitation light, while passing the red fluorescence as
well as the 660 and 780 nm measuring beams for PAR-Absorptivity
(see 9.1.1.10 - 9.1.1.14). A short-pass filter ( < 790 nm), mounted in
a threaded metal ring, protects the CCD-detector against excess nearinfrared radiation contained in ambient daylight. For the optical
properties of the camera it is essential that this filter as well as the
RG 665 are placed between CCD-chip and objective lens (increase of
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COMPONENTS OF THE MAXI-VERSION
effective focal length by plane-parallel glass plates). For other Mseries instruments like the MICROSCOPY-Version and the GFP
MINI-Version this filter has to be removed before mounting the
camera.
Fig. 8: Cosmicar-Pentax objective 12.5 mm focal length, short pass filter and
mounting device (left), distance ring (middle)
3.4
CCD Camera IMAG-K7 and objectives K7-MAX/Z and K7MAX/S
Also the CCD Camera IMAG-K7
(Allied Vision Technologies) can be
used, which features a 1/2" chip with
640 x 480 pixel resolution. The data are
digitized within the camera and
transferred via ethernet interface (GigEVision®) to the PC. Analog to the IMAGK6 camera the sockets for connecting the
ethernet cable and the camera-power-supply cable (round-shaped
connector) are located at the rear side of the camera.
The camera can be equipped either with a standard objective
lens featuring 12 mm focal length (F=1.2 / f=12mm - K7-MAX/S) or
17
CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
a Zoom objective lens with 8 – 48 mm focal length (F=1.0 K7-MAX/Z Fig. 10). Different mounting angles The adapter ring in
the LED-Array Illumination Unit (IMAG-MAX/L) has to be
detached for using the Zoom objective.
Fig. 9: Adapter Ring of the LED-Array Illumination Unit
A 3 mm RG 645 (Schott) color glass filter serves as long-pass filter
for protecting the CCD-chip from blue excitation light, while passing
the red fluorescence as well as the 660 and 780 nm measuring beams
for PAR-Absorptivity (see 9.1.1.10). A short-pass filter ( < 770 nm),
mounted in a threaded metal ring, protects the CCD-detector against
excess near-infrared radiation contained in ambient daylight. For the
optical properties of the camera it is essential that this filter as well as
the RG 645 is placed between CCD-chip and objective lens (increase
of effective focal length by plane-parallel glass plates).
Using the IMAG-K7 camera fluorescence image intensity is
about 50 % of that obtained with the IMAG-K6 camera. While on
one hand it does not allow 4-pixel binning, on the other hand the
applied objective lenses display a higher aperture. Please note that
with the Zoom objective the aperture and consequently image
intensity drop at focal lengths exceeding 30 mm. When properly
adjusted (by provided distance ring and close-up lens) the image
18
CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
remains focused when changing focal length (magnification). Minor
adjustment by the focusing ring may be required.
Fig. 10: CCD Camera with Zoom objective
3.5
Mounting Stand with Eye Protection IMAG-MAX/GS
The Mounting Stand IMAG-MAX/GS provides the standard
means for mounting the powerful LED-Array Illumination Unit at
defined distance to the object, assuring full eye protection of the user.
It features a red perspex hood that absorbs the strong blue light
emitted from the LED-array and at the same time allows to view the
red chlorophyll fluorescence of the sample with bare eyes. The
Measuring Head, consisting of the LED-Array Illumination Unit and
the CCD-camera, is mounted with the help of two clamps on the top
of the Mounting Stand IMAG-MAX/GS.
19
CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
Fig. 11 IMAG-MAX/GS with mounted LED-Array Illumination Unit
IMAG-MAX/L and CCD-Camera
Using this Mounting Stand, the Illumination Unit and the CCDcamera are coupled with each other at a fixed working distance of
18.5 cm between LED-Array and object plane, resulting in
homogenous illumination of an imaged area of 10 x 13 cm. A defined
working distance is important for proper Image Correction (see
9.5.5), which corrects for unavoidable inhomogeneities in measuring
light intensity and camera sensitivity over the imaged area. In
principal, it is also possible to vary the working distance, when the
LED-Array Illumination Unit is mounted independently from the
IMAG-MAX/GS (see Fig. 6). In this case, however, the homogeneity
of the light field may be suboptimal and the user has to take care
about protecting the eyes against excessive light, particularly during
saturation pulses.
20
CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
Fig. 12: IMAG MAX/GS with lifted red-perspex hood showing leaf sample
resting on x-y stage
In standard applications, the sample is resting on an x-y stage
covered with non-fluorescent and non-reflecting black foam-rubber.
The sample compartment becomes accessible after sliding the red
perspex hood upwards, using two flat hands gently pressing against
the two sides. In its fully lifted position the hood is held by two
magnets.
The x-y stage allows to move the sample by maximally 25 and
19 mm in x- and y-directions, respectively. The force stabilizing its
position on the bottom of the Mounting Stand is increased by
magnets mounted at its reverse side.
Note:
During transportation or shipping the x-y stage plate has
to be removed from the sample compartment of the
IMAG-MAX/GS and packed separately within the
Transport Box IMAG-MAX/T to avoid damages.
21
CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
Fig. 13: Bottom part of IMAG MAX/GS with black 96-well microtiter plate
in defined centered sample position
After removing the x-y stage plate, alternatively a multiwell plate
can be placed into the sample plane. A correctly centered position is
defined by two positioning elements (horizontal perspex bar and
metal screw defining right limit). It is recommended to use black
non-fluorescent multiwell plates (e.g. Sigma-Aldrich article no.
M9685). At high sensitivity measurements (high ML intensity, high
Gain), in this application a mirror image of the LED-lamps may be
superimposed on the fluorescence image of the samples contained in
the wells. This phenomenon is due to the fact the blue LEDs emit
some red light, which can be mirrored from the multiwell plate
and/or the water surface of suspension samples via the camera
objective lens onto the CCD chip. If this causes a problem, the red
emission can be removed with the help of the optional Filter Plate
IMAG-MAX/F, which can be mounted in front of the LED-Array
Illumination Unit. Now also clear multiwell plates can be used.
Fluorescence imaging of algae suspensions in 96-well plates can
serve as a powerful tool in ecotoxicology and plant molecular
biology (e.g. screening for mutants).
22
CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
Fig. 14: IMAG MAX/GS, with bottom part being removed, resting on two
spacers to give room for potted plant
Alternatively, it is also possible to remove the bottom part of the
IMAG-MAX/GS for fluorescence imaging of larger samples, like
potted plants. The Mounting Stand can be readily jacked up with the
help of four profile-metal legs with mounting-angles, which can be
screwed to the bottom corners of the Mounting Stand (see Fig. 14).
For this purpose, with the Mounting Stand being turned upside
down, the four nuts first have to be put into the grooves at the bottom
corners. Standard legs with 20 cm length (including mounting screws
and nuts) are delivered with the instrument. With the thus increased
distance to the bottom, the use of a Screw Jack is advantageous,
which allows to move a sample (e.g. potted plant) up/down for
focusing the image at the standard working distance of 18.5 cm. In
this case, the focus of the objective lens should be set for 18.5 cm
working distance (between front of LED-Array and sample) and then
remain unchanged.
23
CHAPTER 3
3.6
COMPONENTS OF THE MAXI-VERSION
Leaf Distance Holder IMAG-MAX/B
Fig. 15: Leaf Distance Holder IMAG-MAX/B with IMAG-MAX/L and
IMAG-K6
Using the Leaf Distance Holder IMAG-MAX/B the illumination unit
with CCD-camera are mounted at standard working distance of 18.5,
resulting in homogenous illumination of an imaged area of
10 x 13 cm of the x-y stage plate.
The assembly of this light weight device is simple. The four black
legs are screwed to the provided screw holes in the bottom corner of
the LED array as well as to the base containing the x-y stage plate.
For convenient sample preparation the x-y stage plate can be taken
off the base by unscrewing the knurled head screws.
24
CHAPTER 3
COMPONENTS OF THE MAXI-VERSION
Warning:
IMAG-MAX/B does not provide eye protection! The user
should avoid looking directly into the LED-Array
Illumination unit.
3.7
Notebook PC IMAG-PC
Operation of the IMAGING-PAM requires a PC with the
following minimum requirements:






processor 1.7 GHz
RAM memory 4 GB
built-in USB interface
built-in ethernet interface GigE-Vision®
built-in DVD/CD-RW drive
operating system Vista, Windows 7 32- or 64-Bit, Windows 8
As operation in conjunction with the IMAGING-PAM
requires 100 % duty cycle of the CPU, an effective ventilating
system is required for cooling the CPU.
The Notebook PC IMAG-PC is delivered with fully installed
software. Depending on the market situation, best value brand-name
devices are chosen which have proven well suited for use in
conjunction with the IMAGING-PAM.
If the instrument has been purchased without PC, the user first
has to install the software (see chapter 6.2)
25
CHAPTER 3
3.8
COMPONENTS OF THE MAXI-VERSION
Adapter IMAG-MAX/GWK
The combined application of IMAGING-PAM and gas exchange
measurements by GFS-3000 provides comprehensive analysis
options. For examples physiological heterongeneities or differences
in genotypes can be visualized in fluorescence images under
changing CO2, O2 or temperature conditions applied by the gas
exchange system. The adapter IMAG-MAX/GWK positions the
MAXI-IMAGING PAM on top of the GWK1 providing eye
protection during the merge of imaging and gas exchange analysis
over a measuring area of 10 x 13 cm.
26
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
4 Components of the IMAGING-PAM MINIversion
The MINI-version of the IMAGING-PAM consists of:
1) Control Unit IMAG-CG with Battery Charger 2120-N
2) MINI-Head (blue) IMAG-MIN/B or
MINI-Head (red) IMAG-MIN/R or
MINI-Head (GFP) IMAG-MIN/GFP
3) CCD Cameras IMAG-K7 or IMAG-K6 with camera objectives
K7- or K6-MIN and mounting sets K7- or K6-MIN/M and K6MIN/FS for use with IMAG-MIN/GFP
4) PC with ImagingWin-software
A useful accessory for leaf measurements is the Leaf Holder IMAGMIN/BK. For simultaneous measurements of gas exchange with the
GFS-3000 the Adapter IMAG-MIN/GFS is available. Also available
for convenient use is a laboratory stands as well as a tripod and a fine
drive tripod adapter for outdoor applications.
4.1
Multi Control Unit IMAG-CG
The same Multi Control Unit IMAG-CG is used for all versions of
the IMAGING-PAM M-series. It was already described in section
3.1.1. in conjunction with the MAXI-version. The LED-Array of the
MINI-Head is plugged into to the MINI-Head connector on the rear
side of the IMAG-CG unit. The Camera cable connector is located
on the front side of IMAG-CG.
The MINI-Head with mounted camera can directly be attached on
top of the Control Unit housing IMAG-CG. For this purpose a wing27
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
screw is provided. Alternatively it can be also mounted on a tripod or
be held by hand using a special grip (see 4.5).
4.2
MINI-Head LED-Array IMAG-MIN/B and IMAG-MIN/R
Fig. 4.1 Top view of MINI-Head IMAG-MIN/B LED-array without
aluminum rods holding the sample-platform
The blue and red versions of the MINI-Head (IMAG-MIN/B and
IMAG-MIN/R) are identical except for the color of the LEDs and the
LED-filters. As blue and red LEDs display different intensity-current
relationships, different PAR-lists apply, that are incorporated in two
different versions of the ImagingWin program (see chapter 10).
The LED-array of the MINI-Head features 12 high-power LEDlamps each equipped with collimating optics, which are arranged in 4
groups of 3 LEDs. Each group is equipped with a short-pass filter
eliminating red light that otherwise would pass the long-pass filter in
front of the CCD-camera and overlap with chlorophyll fluorescence.
In the blue version (IMAG-MIN/B emission peak 460 nm) a bluegreen glass filter (Schott BG39) is used. In the red version (IMAG28
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
MIN/R emission peak 620 nm) a special short-pass interference filter
(< 645 nm) is used. The LEDs are mounted at an angle that is
optimized for obtaining a homogenous light field at the given
working distance. These LEDs provide the pulse-modulated
excitation light and at the same time serve for actinic illumination
and Saturation Pulses. In addition, there are four groups of LEDs
(2x6 and 2x4) providing the pulse modulated light for assessment of
PAR-Absorptivity (see 5.4.1.10 - 9.1.1.14). These LEDs are arranged
in pairs, with each pair featuring a red (660 nm) and a near-infrared
(780 nm) LED. The lenses of these LEDs are removed in order to
obtain homogenous illumination of the sample. While only a
relatively small amount of this light is remitted from the sample to
the CCD-camera, this is sufficient to give good signals, as both
wavelengths can pass the red long-pass filter in front of the CCDchip, in contrast to the filtered excitation light.
The LED-array cable connects to the MINI-HEAD socket at the
rear side of the IMAG-CG Control Unit.
Warning:
Please avoid looking directly into the LEDArray Illumination unit to prevent Eyedamages!
The MINI-Head is designed for a fixed working distance
between camera and sample (7 cm), which is defined by four
aluminum rods with a sample-platform on top of the LED-Array. At
the standard working distance, using the 16 mm lens, a 24 x 32 mm
area is imaged.
All MINI-Head LED-Arrays can be extended with a Leaf Clip
and a Grip Holder (see chapter 4.5).
The MINI-Head LED-Array IMAG-MIN/GFP is described in
chapter 4.4.
29
CHAPTER 4
4.3
COMPONENTS OF THE MINI-VERSION
CCD Camera IMAG-K6 or IMAG-K7
The CCD Cameras IMAG-K6 or IMAG-K7 are described in
chapter 3.4 for the imaging MAXI-version. Both can also be used in
the imaging MINI-version in combination with IMAG-MIN/B and
IMAG-MIN/R. In combination with IMAG-MIN/GFP only IMAGK6 can be used (see chapter4.4.)
For usage in the Imaging MINI-version these CCD cameras need to
be equipped with the objective K6-MIN for IMAG-K6 (F1.4/f = 25
mm) and an 7.2 mm distance ring or K7-MIN for IMAG-K7 (F1.4/f
= 16 mm) with an 4.2 mm distance ring. For both configurations a
3 mm RG 645 (Schott) color glass filter serves as long-pass filter for
protecting the CCD-chip from blue excitation light, while passing the
red fluorescence as well as the 660 and 780 nm measuring beams for
PAR-Absorptivity (see 9.1.1.10). A short-pass filter ( < 770 nm),
mounted in a threaded metal ring, protects the CCD-detector against
excess near-infrared radiation contained in ambient daylight. For the
optical properties of the camera it is essential that this filter as well as
the RG 645 is placed between CCD-chip and objective lens (increase
of effective focal length by plane-parallel glass plates).
Please adjust the objective aperture! Closing the aperture
increases focal depth and decreases light delivery to the
camera (to avoid signal saturation).
The cameras are mounted to the metal holder preinstalled on top
of the MINI-Head LED-Array. The camera side with four screw
holes and the label points to the same direction as the label of the
LED-Array, the camera side with seven screw holes points to the
same direction as the backside of the LED-Array, where the cables
are located and the metal angle bar of the mounting set is mounted.
Fig. 17 displays the mounting positions and required screws for
mounting the camera and the metal angle bar.
30
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
Fig. 16: Camera side with 4 screw holes and label (left) - facing the labelled
LED-Array side, camera side with 7 screw holes (right) facing the
LED-Array backside and the metal angle bar (Fig. 17 A)
5
5
IMAG-K6 GFP/PS II
4+A IMAG-K6 with K6-MIN
4+A
IMAG-K7 with K7-MIN
5
A
1+3
1+2+A
1
2
3
4
5
Fig. 17: Screw indication and camera mounting positions, metal angle bar
including grip of Leaf Holder IMAG-MIN/BK, screw annotation
The metal angle bar of the mounting set K6 and K7-MIN/M (Fig.
17 A) serves for mounting the MINI-Head with camera onto the
IMAG-CG Control Unit, to the grip of the Leaf Holder IMAGMIN/BK or to a tripod. Furthermore the mounting set K6 and K731
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
MIN/M contains a perspex device to unplug the GigE cable from a
mounted CCD camera (see Fig. 18)
Fig. 18: Perspex device to unplug GigE cable from a mounted camera
4.4
IMAG-MIN/GFP with IMAG-K6
For GFP measurements using the MINI-version the IMAGMIN/GFP LED array is needed in combination with the filter slide
IMAG-K6/FS, the 2/3" CCD camera IMAG-K6, the K6-MIN
objective and the K6-MIN/M montage set.
As the LED-array of the other MINI-Heads IMAG-MIN/GFP
features 12 high-power LEDs (emission peak 470 nm) arranged in 4
groups of 3 LEDs and equipped with a short-pass filter (< 500 nm).
These LEDs provide the pulse-modulated GFP excitation light and at
the same time serve for actinic illumination and Saturation Pulses. In
addition, there are four groups of LEDs featuring red (660 nm) and a
32
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
near-infrared (780 nm) LED pairs providing the pulse modulated
light for assessment of PAR-Absorptivity (see 5.4.1.10 - 9.1.1.14).
The LED-array cable connects to the MINI-HEAD socket at the
rear side of the IMAG-CG Control Unit.
The detector filter slide K6-MIN/FS is mounted in front of the 25
mm camera objective K6-MIN as shown in Fig. 19 .
f a
Fig. 19: Standard lens for IMAG-MIN/GFP (focus ring = f, aperture = a),
filter slider in GFP position
Before mounting the objective with filter slide to the IMAG-K6 CCD
camera a 5 mm distance ring needs to be mounted and if IMAG-K6
has been used in another IMAGING-PAM configuration additional
short pass filters (e.g. Fig. 8 left) between camera and objective as
well as other distance rings need to be removed. Afterwards the
IMAG-K6 CCD camera with distance ring, objective and filter slide
is attached to the IMAG-MIN/GFP head using the topmost position
of the metal holder (see Fig. 17).
For special applications it is also possible to use a lens with a wider
angle (16 mm). In this case the camera has to be mounted in one of
the two lower positions (not shown) with the filter slider mounted on
the camera lens subsequently.
With the help of this filter slider the detection filter in front of the
25 mm lens can be exchanged from PSII measurement to GFP
measurement and vice versa.
33
CHAPTER 4
A
COMPONENTS OF THE MINI-VERSION
B
C
Fig. 20: switching of the filter slider between two positions (A – PSII, CGFP position)
The view into the GFP Mini-Head from the sample side (Fig. 20)
explains the two positions of the filter slider.
The filter slider carries two detection filters. The dark red one
(RG665) enables the camera to detect PSII-fluorescence and a
special interference filter for the detection of wavelengths between
500 and 600 nm is used for GFP detection.
34
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
The filter slider can be pushed from left to right (Fig. 20 A to C), to
change the detection filter from PSII-fluorescence to GFP. For
lowering background effects, the GFP Head is shipped with a sample
of a low fluorescent black adhesive film which can be used as a
background layer.
A
B
Fig. 21: possible color modes for the GFP images
35
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
There are different color modes reasonable for the GFP
measurements. Fig. 21 A shows an image in the false color scale
mode of the “Analysis” function (see also chapter 9.1.2.6). Another
option is the black and white mode as in Fig. 21 B. The Description
of the Display Parameters can be found under chapter 9.5.8 (display
parameters) of the manual. Pictures can be exported via the export
function described in chapter 8.2.
Fig. 22: Expanded Color for enhancing GFP images
This example picture (Arabidopsis thaliana with promSUC2 GFP)
has been measured with the following settings:
-
Filter slider in the GFP position
-
MF 8, ML max, Damping 4, Gain on 8
In contrast to the PSII fluorescence, for GFP measurement some
settings must be very high. For this reason long exposure to these
36
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
settings may lead to the bleaching of the sample. Using the
„Analysis“ sliders, the inevitable background can be suppressed.
A
B
Fig. 23: Fo image in the PSII mode (A) vs. Fm image (B)
Since the camera used for the combined GFP-Head is highly sensitive, settings have to be changed between GFP and PSII measurement:
-
PSII fluorescence – ML=1, Gain=2, Damp=1
The basic image fluorescence now should have a value of nearly
0.15, to prevent overflow during the saturating flash. If still overflow
appears, please also use the aperture of the camera lens to lower the
fluorescence signal. (The benefit of doing this is that the depth of
focus is increased which may help to get also bent leaves in focus
without cutting.)
For Absorptivity measurements in the PSII mode no starting values
for red and NIR can be given because these values depend on the
aperture of the used lens. For details read more under chapter 5.8.3
and 5.4.2.1 of this manual.
As for the other MINI-head LED arrays the metal angle bar of
the mounting set K6-MIN/M serves for mounting the MINI-Head
with camera onto the IMAG-CG Control Unit, to the Leaf Holder
IMAG-MIN/BK or to a tripod and can be extended with a Leaf Clip
and a Grip Holder.
37
CHAPTER 4
4.5
COMPONENTS OF THE MINI-VERSION
Leaf Holder IMAG-MIN/BK with Grip Holder
Fig. 24: MINI-Head with CCD camera and grip holder
When the Leaf Holder is delivered together with the MINIversion of the Imaging-PAM, it is already mounted on the MINIHead. A grip holder is provided, which can be fixed to the metal
angle on the camera. Using this grip the MINI-Head can be carried
with one hand and the lever of the leaf clip can be moved up with the
same hand (seeFig. 25).
Fig. 25: Mini-Head with grip holder used in field
38
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
This is particularly useful in field applications. The MINI-Head
may either be carried separately or together with the IMAG-CG, on
which it may be mounted with the help of a wing screw.
Alternatively the Mini-Head may also be mounted on a tripod using
the same metal angle to which the carrying grip can be connected
(see Fig. 26).
Fig. 26: Mini-Head with Leaf Holder mounted on a tripod
When the Leaf Holder is ordered separately from the MINIHead, it has to be assembled by the user. Fig. 27 shows the
components of the Leaf Holder. The following figures (Fig. 28 - Fig.
30) may help to put the various parts together.
39
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
Fig. 27: Components of the Leaf Holder
First the frame (1) is mounted with two screws (2) on the sample
platform of the MINI-Head. Then the clip (3) is fixed with two
screws (4) to the sample platform. While Fig. 28 shows the mounted
clip from the bottom (camera) side, Fig. 29 shows it from the top
side. On the perspex top side of clip the nylon screw (5) is fixed
which holds the two O-rings (6) which function as a spring forcing
together the two parts of the clip (see Fig. 28 and Fig. 29). The Orings may age and then have to be replaced. For this purpose the
spare O-rings (7) are provided. The grip (8) is fixed with the two
screws (9) via a metal angle to the camera, as shown inFig. 30.
40
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
Fig. 28: Leaf clip mounted on sample platform of MINI-Head viewed from
camera side
Fig. 29: Leaf clip mounted on sample platform of MINI-Head viewed from
top side
41
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
Fig. 30: Leaf clip and grip holder mounted on MINI-Head
4.6
Adapter for GFS-3000 (IMAG-MIN/GFS)
The MINI-version of the IMAGING-PAM M-series can be
applied for simultaneous measurements of CO2 gas exchange and
chlorophyll fluorescence. For this purpose the adapter IMAGMIN/GFS is available which replaces the sample-platform of the
standard version. The adapter features a frame with two ball-pins that
allow to click the MINI-Head onto the Measuring Head of the GFS3000. The distance rods carrying this frame are 11 mm shorter than
in the standard version in order to assure identical working distance
from the camera to the leaf sample within the gas exchange cuvette.
42
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
Fig. 31: MINI-Head mounted on Standard Measuring Head of the GFS3000 Gas Exchange Fluorescence System
When the MINI-Head is used in combination with the gas exchange
measuring system, it is controlled by the combined ImagingWin and
GFSWin programs running synchronously on the same PC. The
synchronous operation allows an accurate time assignment of gas
exchange and fluorescence data in a common Report file. While the
actinic illumination for driving photosynthetic electron transport is
provided by the LED-array of the MINI-Head, the switching on/off
of actinic light is controlled via the GFSWin software. Further
peculiarities that are important for the combined operation of MINIHead and GFS-3000 under GFWin and ImagingWin are described in
a separate brochure.
43
CHAPTER 4
4.7
COMPONENTS OF THE MINI-VERSION
ImagingWin software versions for various types of MINIVersion
Upon start of the ImagingWin program the user may choose between
different software versions for the various types of MINI-Heads.
While these versions are practically identical, they feature different
PAR-Lists (found under „Options“ in the software or chapter 10 in
the manual).
Particular Image Corrections (found under „Settings“ or 9.5.5)
apply to different MINI-Heads.
Note:
When the program is started for the first time after installation of the
software, it comes up with a warning that the file for Image
Correction is not found. It is recommended that the user determines
the Image Correction for his particular MINI-Head before starting
serious measurements (see chapter 9.5.5).
With every Measuring Head of the three different correction images
can be stored: Type 1, Type 2 and Maxi or Mini or IMAG L450
and RGB.
For measuring Image Correction please proceed as follows:
• set the optical conditions under which the actual measurements
are going to be done (working distance, focusing position, see above)
• select Type 1, Type 2 or Maxi/Mini/Micro/IMAG L450/RGB
(under Settings/Image Correction)
• in the case of MAXI- and MINI- versions place at least two
layers of white paper (e.g. folded DIN-A4) into sample plane; in the
case of the MICROSCOPY-version the plastic fluorescence standard
• put the image somewhat out of focus to avoid imaging fine
structures of the white paper tissue or dust etc. on the surface of the
fluorescence standard
•
44
press the measure button (under Settings/Image Correction)
CHAPTER 4
COMPONENTS OF THE MINI-VERSION
The measured correction image will be saved until it is
overwritten by a new measurement. The correction images will
remain valid as long as the same optical parameters apply (LED
Illumination Unit, working distance, focusing position, camera
objective lens, microscope objective lens).
45
CHAPTER 5
COMPONENTS OF THE MICROSCOPY-VERSION
5 Components of the IMAGING-PAM
MICROSCOPY-version
The MICROSCOPY-version of the IMAGING-PAM consists of:
1) Control Unit IMAG-CG with Battery Charger 2120-N
2) CCD Camera IMAG-K6 (2/3")
3) Modified Zeiss Axio ScopeA.1
4) LED Modules as:
Microscopy LED Lamp (blue) IMAG-L470M or
Microscopy LED Lamp (red-orange) IMAG-L625M or
Microscopy LED Lamp (UV-A) IMAG-L365M or
Red-Green-Blue Microscopy LED Lamp IMAG-RGB
5) PC with ImagingWin-software
Operation of the MICROSCOPY-IMAGING-PAM requires an
epifluorescence microscope. For this purpose relatively simple
microscopes with short excitation pathways are suited. Most
essential components for optimal image qualities are high aperture
objectives and a suitable video adapter. While the IMAG-K6 camera
features a 2/3" CCD chip, it is recommended to use a 0.5x video
adapter for 1/2" chips, in order to obtain a more intense fluorescence
image.
The Zeiss Axio Scope.A1 microscope may be particularly
recommended for use in conjunction with the MICROSCOPYversion.
The features of these components will be described briefly in the
following subsections.
46
CHAPTER 5
5.1
COMPONENTS OF THE MICROSCOPY-VERSION
Multi Control Unit IMAG-CG
The same Multi Control Unit IMAG-CG is used for all versions
of the IMAGING-PAM M-series. It was already described in section
3.1 in conjunction with the MAXI-version. The cable of the
MICROSCOPY LED Lamps IMAG-L470M or IMAG-L625M is
connected to the MINI-Head socket at the rear side of the control
unit. The cable of the Red-Green-Blue Microscopy LED Lamp
IMAG-RGB is connected to the RGB-Head socket at the rear side of
the control unit. The rear side also features a CAMERA-socket to
which the Camera cable has to be connected. This cable is used to for
trigger signal and to power the IMAG-K6 camera.
5.2
CCD Camera IMAG-K6
The CCD Camera IMAG-K6 as described in chapter 3.3 features
a 2/3" chip with 1392 x 1040 pixels and 4-pixel-binning, resulting in
fourfold image intensity for the 640 x 480 pixel displayed on the
monitor screen.
If the IMAG-K6 has been used in another IMAGING-M
application. Please take off the objective, filters and the distance
ring. The IMAG-K6 camera can now be connected on top of the
phototube of the Axio Scope.A1 via video adapter. 0.5x adapter for
1/2" CCD cameras (Zeiss; 416112-0000-000, Fig. 32).
47
CHAPTER 5
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Fig. 32: Camera adapter for connecting Axio Scope.A1 with IMAG K6
Fig. 33: Zeiss camera adapter mounted on Axio Scope.A1
The camera IMAG-K6 (not shown) can be screwed onto the Cmount adapter shown in Fig. 33. Afterwards the camera needs to be
connected to the camera port of the IMAG-CG multi control unit as
well as to the computer via Gigabit Ethernet.
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COMPONENTS OF THE MICROSCOPY-VERSION
The user should get familiar with the
switch which allows switching between
binocular or phototube / photomultiplier pathways (see Fig. 34).
Fig. 34: Pathway switch
5.3
Furthermore the halogen transmitted
light lamp should be switched off
during epifluorescence measurements to
avoid actinic illumination of the sample.
Axio ScopeA.1 Epifluorescence Microscope
Fig. 35: MICROSCOPY-IMAGING-PAM based on Zeiss ScopeA.1
Epifluorescence Microscope
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The MICROSCOPY-IMAGING-PAM is readily adapted to the
Zeiss Axio ScopeA.1 microscope giving excellent fluorescence
images. If a complete IMAGING-PAM MICROSCOPY Version has
been purchased, some parts are already mounted to make the first
setup a bit easier. For safe shipping purposes some components had
to be detached.
Existing Zeiss Axio ScopeA.1 microscopes can be adjusted to the
MICROSCOPY-IMAGING-PAM on request.
Please note that openings in the microscope parts are sealed by
stickers or caps that have to be removed before mounting the parts
The Axio Scope.A1 has a special port that can carry up to four
LED modules which are available in ten different wavelengths
starting with 380 nm up to 625 nm. For the standard PAM
application we are recommending the 625 nm red LED module or the
470 nm blue light version (not suitable for the measurement of
cyanobacteria). More Information on the Zeiss LED modules can be
found in chapter 3.4.9 of the Axio Scope.A1 manual or in the internet
on www.Zeiss.de. The LED modules offer very homogeneous
illumination of the measured area and are modified for PAM
applications with additional filters by Walz.
When other wavelengths, than the recommended ones, shall be
applied for special measurements, please contact Walz for
recommendations on appropriate filter combinations.
Important parts required for optimal performance are the microscope
objective lenses. High aperture lenses like the Zeiss Fluar 10x/0.5,
Fluar 20x/0.75 and Fluar 40x/1.30 Oil are recommended. In this
context, it has to be considered that a high aperture enhances image
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COMPONENTS OF THE MICROSCOPY-VERSION
intensity two-fold by increasing excitation intensity as well as
fluorescence collection. The image intensity with a Zeiss Fluar
20x/0.75, for example, is 6.6 times higher than with a Zeiss Apoplan
20x/0.45. In view of the fact that image intensity is the limiting
factor in MICROSCOPY-PAM applications, an investment in high
aperture lenses should have high priority.
5.3.1 Reflector Modules
In complete IMAGING-PAM MICROSCOPY instruments
necessary filters of the reflector modules are already mounted.
In case the reflector module is purchased as a separate part, please
follow the instructions below. The correct orientation of the beam
splitter filter is essential for the correct functioning of the
IMAGING-PAM system.
It is required to mount the reflector modules into the turret of the
Zeiss Axio Scope.A1 as described in chapter 5.3.3. When using more
than one LED module, please note that each LED module needs its
own reflector module (chapter 3.1.6 Fig 3-9 and 3.4.6 Zeiss manual).
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5.3.2 Assembling of beam splitter and filters

B
Zeiss
Zeiss
Fig. 36: The reflector module – mounting the beam splitter
A) For opening the reflector module, loosen two screws (A/1). The
emission part (A/2) can now be detached from the excitation part
(A/3) by a turn around the lower angle. B) Tilt the excitation part on
top and lift the emission part out of the holding fixtures. A spring box
(B/3) holds the beam splitter filter. Take care that the mirrored side of
the beam splitter is pointing upwards.

LED
B
Zeiss
Fig. 37: Reflector module with filter configuration
52
Zeiss
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COMPONENTS OF THE MICROSCOPY-VERSION
A) The arrows in Fig. 37/A1 mark the path of the illumination beam
or the imaging beam seen from the side. B) lists the different filters
and parts that may be needed. The detector filter B /2 (RG665), a
filter for the excitation light source (B /5 - normally not needed). B
/6 is the mounting tool for the adapter rings (B /1). The beam splitter
filter (A /3) is already mounted in this figure (see Fig. 37/B).
Zeiss
Note:
the reflecting (coated)
side of the color splitter
has a tapered edge or
corner.
Fig. 38: Labeling the color splitter
When purchasing the filters separately, independently of the
original Zeiss reflector module frame (424931-0000-000), they have
to be mounted into the reflector module FL according to the
description in this chapter. The red filter RG665 (25 mm in diameter)
is inserted on top of the reflector module and held by the adapter ring
1 (Fig. 37/B), so that the filter will show towards the camera of the
IMAGING version.
The rectangular beam splitter filter is mounted in a 45° angle (Fig.
37/A3). The mirrored side has to show towards the LED light source.
The coating faces outward (in relation to the reflector module) in the
direction of the excitation filter (Fig. 37/B5 – not used in the
standard setup).
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5.3.3 Mounting of the reflector module
The mounting of the reflector module into the Zeiss Axio Scope.A1
is described in Fig. 39.
Zeiss
Fig. 39: Changing the reflector module in the upper stand FL-LED
In the front of the microscope a grey plastic cover, behind which the
turret for the reflector modules can be found, can be pulled off in
forward direction for mounting the reflector modules in the reflector
turret. It is just held in place by magnets.
Remove the cover cap (indicated with 1 in Fig. 39) in front of the
filter turret to get access to the reflector module ports.
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Fig. 40: Mounting the reflector module
The reflector module is inserted after turning it by 180° around its
vertical axis (the reflector module is mounted with its excitation filter
side Fig. 37/A7 facing to the front). It shall be inserted carefully into
the upper spring elements. Then engage it firmly by gently pressing
it down into the turret.
When switching to another LED light source, also the reflector
module is switched. Please make sure that the numbers of the LED
module positions correspond with the numbers of the reflector
modules in the turret of the Axio Scope.A1.
A grey circle (a) in Fig. 40 marks the switch that normally regulates
the LED modules. Since the LED modules for PAM use will be
connected via the top cover of the microscope with the central
control unit of the imaging system (IMAG-CG), this switch is not
active anymore (see chapter 5.4.3).
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5.4
COMPONENTS OF THE MICROSCOPY-VERSION
LED Modules
There is a wide choice of wavelengths available for the Zeiss LED
modules so that experienced users can easily modify their
epifluorescence system by additional light sources with various
wavelengths. LED modules might need to be adapted as described in
this chapter. Also the filters used in the reflector module might be
necessary to adapt (see chapter 5.3.3)
5.4.1 Adjustment of brightness by grey filters
Since the original LED modules from Zeiss are far too bright for
PAM purposes (especially for the measuring light) neutral grey filters
have to be used.
A set of these filters are provided with each LED module purchased
from Walz (Y = 6,6; 13,7; 23,5 and 51,2). The darkest ones are
already used for dimming the LED. Two more neutral grey filters are
provided for fine adjustment
when other magnifications like
the standard 20 x Fluar lens
(Zeiss) are used. The higher the
magnification used, the smaller
the illuminated spot of the
imaged field, which means that
there
is a good chance to increase
Fig. 41: LED-module filter tool
measuring light intensity on the
sample plane too much. In this case it might be necessary to change
the filter composition in front of the LED module by using the
provided tool shown in Fig. 41. The side pointing upwards is used
for mounting the filters of the LED modules.
Zeiss LED modules are shipped together with a set of filters:
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The red LED module IMAG-L625M
comes with a set of grey neutral
density filters that can be used to adapt
the emitted light to different
magnifications.
The
additional
KPF647,5 filter cuts off unwanted
wavelengths and shall be mounted as
last filter in front of the ND filters.
With the blue LED module
IMAG-L470M also four ND filters are
shipped. With this unit no additional
KPF filter is necessary.
The 365 nm module comes also with light grey Lee filters. These
have a transmission of 40% each in the range of 365 nm so that these
filters can also be used to adapt the intense ultraviolet irradiance. The
ND filters shall be mounted between the lamp and the BG39 filter
which is always recommended to be used as last optical filter
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This lamp also needs a special reflector module (Beam-splitter 395
nm Zeiss 446431-0011-000). Make sure not to intermix this part
with other colorsplitter modules.
Since this is no standard part, please inquire at Walz.
5.4.2 Integration of LED modules into Axio Scope.A1
The integration of LED modules in the Axio Scope.A1 is shown in
Fig. 42.
Zeiss modified
Fig. 42: Changing the LED module in the upper stand part FL-LED
-
58
Lift the covering cap (Fig. 41 indicated with number 1) off
the upper stand part.
Remove the connection plug of the LED module to be
changed number 2 and 3 (Fig. 41) from the corresponding
slot and pull the LED module out of its socket.
CHAPTER 5
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-
-
COMPONENTS OF THE MICROSCOPY-VERSION
Insert the new LED module into the socket and plug the
cable into the corresponding slot. No further adjustment is
necessary.
As the LED circuit is mechanically coupled to the reflector
turret, it is necessary to make sure that LED module and
fluorescence filters on the corresponding reflector turret
position are compatible.
For better operation, the positions of the LED module and
those in the reflector turret are numbered.
In the case that the Axio Scope.A1 shall also be used for further
epifluorescence applications beside PAM measurements, the
electrical connections shown in Fig. 42 (cables a and b connecting
the LED modules with the IMAG-CG unit via rotary switch
indicated with number 5) have to be modified depending on the
number of LED modules used for each application. Please ask for
technical assistance at the Heinz Walz GmbH if a combined
application is intended.
Please Note: If cables a and b in Fig. 42 are connected the switch for
the epifluorescence lamps on the right side of the microscope is not
active (Fig. 40 grey circled a).
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5.4.3 Connecting LED modules with IMAG-CG
For the use together with IMAGING-PAM MICROSCOPY version
the LED modules have to be connected with the central control unit
IMAG-CG. The provided connection cable for the LED modules has
to be connected with the 3-pin connector (Fig. 42 number 6) on the
cover cap of the Zeiss Axio Scope.A1 and with the 6-pin “MINIHead” connector on the IMAG-CG control unit.
5.4.4 Switching LED modules for measurements
Switching from one LED module to another is done by turning the
filter wheel on the front of the reflector turret. Additionally the rotary
switch on the right side of the covering cap (Fig. 41 indicated with
number 5) has to be set to the corresponding number of the reflector
module chosen, so that the LED module is engaged for the
measurement.
5.4.5 IMAG-RGB
Fig. 43: IMAG-RGB Microscopy LED Lamp
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The Red-Green-Blue Microscopy LED Lamp (in contrast to the
IMAG-L470M or IMAG-L625M) features its own LED drivers. It is
connected to the RGB-Head socket at the rear side of the IMAG-CG
control unit. Optically it is connected with the epifluorescence
microscope via a fluid light guide (100 cm length, 3 mm ). The
IMAG-CG control unit provides the power and the trigger signals for
driving the 3 types of LEDs. Special pigtail-LEDs exclusively
developed for PAM fluorometry are used. These LEDs give
exceptionally high light intensities at the exit of 1 mm  plastic
optical fibers. The fibers of 7 LEDs (2x Red, 3x Green, 2x Blue) are
put together resulting in a 3 mm  bundle, to which the 3 mm 
fluid light guide connects. The latter serves for thorough mixing of
the three colors and for carrying the light to the epifluorescence
microscope excitation entrance port.
a
b
insert
fine
Fig. 44: Fluid Light Guide with Adapter Endpiece for connecting to
excitation entrance port of Epifluorescence Microscope
The microscope side of the fluid light guide features a metal tube
adapter with an aspherical lens that fits to the LED module socket of
the Axio Scope.A1
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-
Lift the covering cap (Fig. 41 indicated with number 1) off
the upper stand part.
- Remove the connection plug of the LED module to be
changed number 2 and 3 (Fig. 41) from the corresponding
slot and pull the LED module out of its socket.
- Insert the fluid light guide metal tube into the socket.
- Close the covering cap.
- Insert the fluid light guide through the whole in the covering
cap into the fluid light guide metal tube.
As the LED circuit is mechanically coupled to the reflector
turret, it is necessary to make sure that LED module and
fluorescence filters on the corresponding reflector turret position
are compatible.
Fig. 45: Schematic presentation IMAGING-PAM MICROSCOPY-version
with IMAG-RGB
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6 How to get started
While some parts of this section specifically refer to the MAXIImaging-PAM, most information applies to all versions of the
Imaging-PAM M-series. Specific information on MINI-, and
MICROSCOPY-versions is presented in sections 4 - 1.1.
The IMAGING-PAM is readily set up and its basic operation is
quite simple. The following sub-sections explain how to put the
system together and how to install the software on the PC. Also some
simple measurements will be described, which may help the user to
become acquainted with the instrument.
6.1
Connecting the cables
There is a total of 3 (in case of MAXI-version 4) cables to be
connected:
1) Camera cable between the CCD Camera and the Control Unit
IMAG-CG (front side)
2) GigE ethernet cable between Camera and PC
3) LED-Array cable connecting to the Control Unit IMAG-CG

MAXI-version: IMAG-MAX/L or IMAG-MAX/LR cable to
MAXI-HEAD socket (front side), and the second LEDArray cable connecting to the separate POWER-SUPPLY
with the red plug to the red (+) socket and the black plug to
the black (-) socket

MINI-version: IMAG-MIN/B, IMAG-MIN/R or IMAGMIN/GFP cable to MINI-HEAD socket (rear side)

MICROSCOPY-version: IMAG-L625M and IMAG-L470M
is connected via MINI-Head cable from the 3-pin connector
(Fig. 42 number 6) on the cover cap of the Zeiss Axio
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Scope.A1 to the 6-pin “MINI-Head” connector (IMAG-CG
rear side), the IMAG-RGB is connected via IMAG-RBG
cable to RBG-HEAD socket (IMAG-CG rear side)
Notes:
All cables should be connected prior to switching on the
Control Unit, the separate POWER-SUPPLY and the
PC is started.
Generally, always first switch off the external POWERSUPPLY before exchanging LED-Array cable.
Never have more than one Measuring Head (MAXI-,
MINI- or RGB-) connected at the time.
6.2
Software installation
All software required for operating the IMAGING-PAM already
is installed, when the Notebook PC IMAG-PC was purchased
together with the instrument. Otherwise the user can install the
required software as outlined in the following sub-sections.
6.2.1 Installation and Starting of ImagingWin
The ImagingWinGigE software is delivered together with the
instrument in form of a CD-ROM. For installation of ImagingWin
this CD-ROM is put into the CD-drive of the PC which is going to
be used in conjunction with the IMAGING-PAM. Installation occurs
automatically (Autostart). If the Autostart function is not active under
Windows, the Set-up file has to be started manually from the CD. A
program icon (ImagingWinGigE) and a link to the ImagingWinGigE
Folder are automatically installed on the Desktop.
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The ImagingWinGigE folder contains all files required for
operation of the IMAGING-PAM and also the Data-directories for
the various types of measuring heads.
For updates of the ImagingWinGigE software update setup files
can be downloaded from the Walz website (www.walz.com). Please
note that the Data directories and all system settings are not affected
by the Update. After clicking "Install" the Windows "Hardware
Installation" may give a warning which, however, can be ignored by
clicking "Continue Anyway". The installer will automatically setup
necessary camera drivers. After installation the software can be
started by clicking on the desktop icon.
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6.2.2 Installation of camera driver
If, for some reason, the previously installed Camera driver got lost,
the re-installation can manually be done by opening the
Allied_Vision_Technologies_GigE_Viewer
located
in
the
ImagingWinGigE folder.
6.3
First steps and examples of routine measurements
After the IMAGING-PAM is set up, all
cables are connected and all software is installed
(see 6.2), first measurements can be carried out
in order to become acquainted with the
instrument. In the following description, use of
the MAXI Measuring Head with the LEDArray Illumination Unit IMAG-MAX/L in
conjunction with the Mounting-Stand IMAGMAX/GS is assumed. Before starting the
program, power should be switched on at the
Control Unit IMAG-CG (via POWER-switch on the front), as well
as at the External 300 W Power Supply (via switch at the rear side).
When the program is started by clicking the ImagingWinGigE.exe
start icon, a selection window appears.
If one of the other Imaging systems (MICROSCOPY- or MINIversion) is used you will be asked to make choice for, for example,
the light color. It is important to chose the right color and head,
because the LEDs may be damaged otherwise.
After selection of the Measuring Head and confirmation by OK,
the Maxi button should be checked and confirmed by O.K. Then the
pulse-modulated fluorescence measuring light is automatically
switched on. Fluorescence is measured with relatively weak
measuring light pulses at low repetition rate (ca. 1 Hz). This weak
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measuring light does not cause significant changes in the given state
of a leaf sample. The PC monitor screen shows the Image window
on which after program start the image of the fluorescence
parameter Ft is displayed. The Ft-image is black as long as no leaf
sample is present. When a leaf sample is placed on the x-y stage
plate, at the given slow rate of measuring light pulses the Ft-image
slowly appears on the screen. In order to arrange a defined position
of the sample within the field of view and to focus the image, it often
is advantageous to switch from Fluorescence-imaging to nearinfrared (NIR)-imaging by selecting Live Video (at the right hand
side of the Image window). Now NIR-measuring light pulses with
which image changes can be followed. The NIR light pulse
frequency is adjustable in the Live Video window.
The image can be focused by turning the adjustment ring of the
objective lens. After having focused the image using NIR-light, the
Live Video window must be quit by clicking "Close" or the exit box
in the upper right corner. Then the system returns to the
Fluorescence measuring mode, displaying the focused Ft-image.
Fig. 46 shows a PC screen shot of a typical fluorescence image of a
leaf under the described conditions.
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Fig. 46: PC screen shot of Ft-image of leaf
In the center of the screen by default a circular area is defined as
so-called Area of Interest (AOI). The Ft values of all pixels within
this area are averaged and the averaged value is shown in the little
red box close to the AOI. Additional AOIs can be defined by the user,
with various shapes and sizes (via the AOI box at the right hand side
of the screen). At the bottom of the image area the false color code
bar is displayed, with the colors encoding for numerical values
between 0 (corresponding to black at the left edge) and 1
(corresponding to purple at the right edge).
So far the IMAGING-PAM has been monitoring fluorescence
yield, but no actual measurement was carried out yet. With the
IMAGING-PAM, just as with most other PAM fluorometers, a
"measurement" means the assessment of photosynthetic parameters
by fluorescence quenching analysis with the help of a saturating light
flash (Saturation Pulse). For determination of so-called quenching
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coefficients, measurement of the minimal and maximal fluorescence
yield of a dark-adapted sample is important. Dark-adaptation does
not have to be strict. Actually, in most cases a few minutes adaptation
to low light conditions are sufficient for serving this purpose.
Warning:
It is recommended that the eye protecting red perspex hood
is slid down before a saturation pulse is given.
The fluorescence intensity excited by the Saturation Pulse is so
high that it can be readily seen by the bare eyes through the red
perspex hood, which absorbs the much stronger blue light, which
would be harmful for the eyes. The "dark fluorescence
parameters" can be assessed by an Fo, Fm measurement.
The corresponding push button is at the bottom of the screen,
together with various other elements for system operation.
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Fig. 47: PC screen shot of Image window following Fo, Fm determination
with the Fv/Fm image being selected and various types of AOI
being defined
In Fig. 47 the Image window following Fo, Fm determination is
shown, when the Fv/Fm-image is selected. Fv/Fm reflects the
maximal PS II quantum yield of a dark-adapted sample. With the
given leaf sample, Fv/Fm is distributed quite homogenously over the
whole leaf. It may be noted that after Fo, Fm determination the Fo,
Fm button is not accessible anymore and that instead the New
Record button has become accessible. The Fo, Fm determination
will remain valid until a New Record is started. All F and Fm' values
measured in conjunction with the help of Saturation Pulses (triggered
via the SAT-Pulse button) are compared with Fo and Fm and
consequently the quenching parameters are calculated. The user may
apply some Saturation Pulses and experience how the images of the
various parameters (e.g. F, Fm', Yield, qP and qN) change with
preillumination.
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When the sample is illuminated, the effective quantum yield is
decreased, as PS II reaction centers partially close (decrease of
photochemical quenching) and energy dissipation into heat increases
(increase of non-photochemical quenching). Actinic illumination can
be started by checking the AL box. Then the PAR box shows the
PAR-value of the incident light. For assessment of fluorescence
parameters during actinic illumination, a Saturation Pulse can be
applied using the SAT-Pulse button. Fig. 48 shows an image of
effective PS II quantum yield, Y(II), measured with the help of a
Saturation Pulse applied after 2 min illumination at 81 µmol quanta
m-2s-1.
Fig. 48: Y(II) image assessed after 2 min illumination at 81 µmol quanta
m -2s -1
This measurement reveals some heterogeneity in the lowering of
quantum yield by illumination in different parts of the leaf. It is
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generally observed that differences in photosynthetic efficiency can
be distinguished best when actinic light is applied, which puts some
pressure on the limiting steps of the overall process, such that
electrons accumulate at the acceptor side of PS II.
Considerable heterogeneity is also displayed by nonphotochemical quenching (expressed by the fluorescence parameters qN,
see also 9.1.1.15) as illustrated in Fig. 49
Fig. 49: Image of the coefficient of nonphotochemical quenching qN
measured 2 min after onset of illumination at 81 µmol quanta m-2–1
The light induced changes in fluorescence parameters are highly
dynamic. When a dark-adapted sample is illuminated, fluorescence
yield first rises and then drops again (dark-light induction curves,
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Kautsky effect). Saturation Pulse quenching analysis reveals that
characteristic changes in quantum yield (YII) and nonphotochemical
quenching (qN) accompany the changes in fluorescence yield. The
Kinetics-window serves for the study of such dark-light induction
phenomena. It is opened by clicking the Kinetics register card at the
top of the screen. For the recording of induction kinetics at least one
AOI has to be defined. Then the recording of an Induction Curve
(Ind.Curv.) can be started (click “Start”).
Fig. 50: Kinetics window showing Induction Curve. Two AOIs are
selected, for which the averaged pixel values of Fm', Y(II), qN and
Ft are displayed.
Under ImagingWin the recording of an Induction Curve
constitutes a New Record, which is first stored in a buffer memory.
It later can be permanently saved on hard disk. An Induction Curve
recording normally starts with an Fo, Fm measurement, on the basis
of which the quenching coefficients are calculated. When the
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recording of the Induction Curve is terminated, ImagingWin quits the
Measure-mode (green check box inactive) and enters the Viewmode, which allows to look at the recorded data. During the course
of an Induction Curve a vast amount of information was stored,
which can be analyzed at any time after the recording. Analysis is
also possible in the Off-line mode, i.e. without the IMAGING-PAM
being connected to the PC. For each Saturation Pulse the images of
the various fluorescence parameters were captured. These images can
be viewed by returning to the Image-window, where the desired
parameter can be selected. When Go is activated, the consecutive
images are shown like in a movie, starting with the data set
corresponding to the Fo, Fm determination. The Go Speed can be
modified under Settings (click the corresponding register card).
Images can also be selected manually after deactivating Go and
clicking with the cursor into the box to the left where each mark
corresponds to a data set associated with a Saturation Pulse. The
current number is also shown in a separate box. In the View-mode
the data can be stored in form of a so-called PAM Imaging (pim)
file on hard disk. Individual images can be also exported in form of
TIFF or JPEG files.
Dark-light Induction Curves give important information on
various steps of the complex photosynthetic process and allow to
identify the site of a possible limitation, e.g. induced by a stress
parameter. The IMAGING-PAM allows to apply this tool with high
reproducibilty using pre-programmed Standard Induction
Curves. The Induction Curve parameters, like Actinic Light
Intensity, time interval between Saturation Pulses and duration of
illumination can be defined by the user under Settings.
Another standard tool for assessment of photosynthetic
parameters by Saturation Pulse quenching analysis are recordings of
Rapid Light Curves (more briefly also called Light Curves). For
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measurement of a Light Curve the user has to return to the Measure
Mode and click the Light Curve register card. While the recording
of a pronounced Induction Curve is favored by previous dark
adaptation, the opposite is true for the recording of a Light Curve,
which should not be dominated by induction effects. Therefore, a
Light Curves can be measured best shortly after an Induction Curve
with the same sample. While the Light Curve is running, one can
either follow the development of the curve on the Light Curve
window or look at the changing images of e.g. Y(II) or qN. The
Light Curve starts with an Fo, Fm determination which, however,
formally is correct only, if the sample was dark-adapted. If a Light
Curve is recorded after an Induction Curve using the same sample,
the previously measured Fo, Fm values may be retained, provided
there was no change in the position of the sample.
Fig. 51: Light Curve window showing the Light Curves of two AOIs, for
which the averaged values of the ETR parameter (relative apparent
electron transport rate) are displayed.
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In Fig. 51 a Light Curve recording of the ETR-parameter is
shown. ETR is a relative measure of the apparent electron transport
rate. It initially shows an almost constant slope and saturates at high
light intensities, in analogy to conventional light response (PI)
curves. It has to be kept in mind, however, that PI-curves are
measured with much longer adaptation times at each intensity step.
The original definition of the ETR-parameter assumes a uniform
absorption of incident light over the whole sample area:
ETR = Yield x PAR x 0.5 x Absorptivity
The Absorptivity parameter describes the fraction of incident
light which is absorbed. The factor 0.5 takes into account that only
half of the absorbed quanta is distributed to PS II (under steady state
conditions). In most studies carried out with standard PAM
fluorometers, like the PAM-2100 or MINI-PAM, it has been assumed
that Absorptivity amounts to 0.84, which is the mean value for a
large number of normal, healthy green leaves determined with the
help of an Ulbricht Sphere.
The IMAGING-PAM offers a special routine for measuring
PAR-Absorptivity images by comparing the remission images of
diffuse red and NIR radiation, which is emitted by the same LEDRing-Array as the blue fluorescence measuring light. Despite of its
simplicity, this routine functions surprisingly well, provided the
intensity of the red light was appropriately adjusted with respect to
the NIR light. A PAR-Absorptivity measurement is started by
clicking the Measure Abs. button. First an NIR-remission image
and then a R-remission image is measured, the Absorptivityparameter is automatically calculated pixel by pixel according to the
equation Abs. = 1 - R/NIR and the Abs.-parameter is displayed on
the Image-window, as illustrated in Fig. 52
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In case an Absorptivity measurement has been made prior the
measurement, the actual measured Abs values will be taken for the
calculation of the photosynthesis parameters like ETR.
Any pigment that absorbs Red more than NIR light, i.e. generally all
photosynthetically active pigments, will decrease R with respect to
NIR, thus decreasing R/NIR and increasing the derived Abs.-value.
On the other hand, pigments that absorb Red and NIR light similarly,
as e.g. necrotic spots, will not cause R/NIR to deviate substantially
from unity and thus Abs. will remain close to zero. Notably, a white
and a black piece of paper give similar Abs.-images with pixel-values
close to zero. This illustrates that the Abs. parameter does not assess
general Absorptivity of a sample for visible light, as sensed by the
human eye, but rather specifically the Absorptivity of
photosynthetically active light. Therefore, the Abs. parameter as
determined by the IMAGING-PAM may be considered a close
estimate of PAR-Absorptivity. This approach is based on the
empirical fact that pigments that contribute to the absorption of PAR
do not show significant absorption bands in the near-infrared (NIR)
spectral region. On the other hands, pigments that absorb NIR are
likely to also absorb Red light.
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Fig. 52: Image of PAR-Absorptivity determined by the Measure Abs.
routine
In the example of Fig. 52 Absorptivity is distributed quite
homogenously (pixel values around 0.9) over the whole leaf area. In
other cases substantial heterogeneities can be observed, e.g. induced
by viral or fungal infections and stress induced damage which leads
to formation of necrotic spots.
With information on PAR-Absorptivity, it is possible to calculate
a relative apparent rate of photosynthetic electron transport:
PS = Yield x PAR x 0.5 x Abs.
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This parameter is calculated by ImagingWin and can be selected
on the Image-window. As all imaged parameters have to be
normalized to values between 0 and 1 (for the sake of a uniform false
color scale), the calculated PS-values are divided by the expected
maximal rate, the preset value of which is 50.
Fig. 53: Image of the relative apparent rate of photosynthetic electron
transport as measured by the PS/50 parameter
The PS/50 image displayed in Fig. 53 reveals that a
homogeneously green looking leaf may show distinct heterogeneities
in photosynthesis.
These first measurements on one hand demonstrate the simplicity
of measurements with the IMAGING-PAM and on the other hand
give a first impression of the vast potential of this tool for assessment
of photosynthetic parameters. This introduction should enable the
user to get acquainted with the instrument and to start carrying out
own experiments. For quantitative work some more information may
be required. In the following Chapter 7 the numerous functions and
features of the ImagingWin software are described systematically in
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more detail. Unavoidably there will be some overlapping with the
information given in this Chapter 6.
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7 ImagingWin
Except for the POWER on/off switch on the Control Unit and the
External 300 W Power Supply, the MAXI-IMAGING-PAM is fully
operated via PC using the ImagingWinGigE software. Fig. 54 shows
the user surface of ImagingWinGigE after start of the program, as
seen on the PC monitor screen.
Fig. 54: User surface of ImagingWinGigE after start of the program
The screen is divided into three parts. A topmost part, containing
the menu bar (see chapter 10). A major upper part, the content of
which changes depending on the particular window selected by the
various register cards 9 (Image, Kinetics, Light Curve, Report,
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Settings and High Sens, chapter 9:) and a bottom part, which relates
to system operation (like saving data, starting measurements etc.)
remains unchanged when different windows are selected (chapter 8).
1) After start of the program, the upper part of the screen by default
shows the Image-window. The other windows can be installed
by clicking the corresponding register cards. The various
windows will be explained in detail in separate sections below.
2) At the bottom of the screen different types of functional elements
essential for operation of the IMAGING-PAM are located:




the elements at the left side relate to the recorded data
(viewing, saving, opening and export of data)
in the middle the functional elements are located which serve
for defining a new recording (New Record; Fo, Fm;
Measure)
the elements to the right relate to the various types of light
sources (PAR, ML, AL, Ext, SAT-Pulse, AL+Y, Clock)
the remaining elements on the right side are for operating the
ImagingPam using script files (Load, Run)
3) The top of the screen locates the menu File, Edit, Options, AlList, Recalc and Transect.
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8 IMAGINGWIN - System Operation
8.1
Definition of New Record
8.1.1 Fo, Fm
The Fo, Fm-determination is of central importance
for recordings with the IMAGING-PAM. Only
after appropriate determination of Fo and Fm with
a more or less dark-adapted sample, the
consequently measured values of the fluorescence parameters qP, qN
and NPQ will be meaningful. All data recorded after an Fo, Fmdetermination are stored as one "Record" in a current buffer memory
(see below) and eventually may be saved as a PAM Image (PIM)file. Please note that upon Fo, Fm determination all data previously
stored in buffer memory will be erased. Therefore, the user is asked:
"Save previous Record?" If this question is answered with "No",
the previously recorded data irrevocably are gone. Upon start of a
Kinetics recording (see 9.2) or Light Curve recording (see 9.3) the
user is asked : "Do you want to keep the previously recorded Fo,
Fm?" During a running Record no Fo,Fm-determination is possible.
Fo- and Fm-images, which can be selected on the Image-window, are
prerequisite for calculation of images of Fv/Fm and of the quenching
coefficients qP, qN and NPQ. The Fv/Fm image not only defines the
maximal PS II quantum yield, but also serves for definition of the
sample limits. This definition is applied for noise suppression outside
the sample limits in Y(II) images.
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8.1.2 New Record
Upon start of a New Record previously recorded
data stored in the buffer memory are erased in
order to make room for the new data. Therefore,
the user is asked: "Save previous Record?" If
this question is answered with "No", the previously recorded data
irrevocably are gone. While normally, a New Record is started by an
Fo,Fm determination, it is also possible to keep the previously
determined Fo, Fm values (see above). It is also possible to carry out
measurements by application of Saturation Pulses without previous
Fo, Fm determination. In this case, however, no quenching
coefficients can be calculated and also the noise suppression based
on the Fv/Fm image (see above) does not work. A later Fo, Fmdetermination within a running Record is not possible. As soon as a
new Fo, Fm determination is carried out, a New Record is started.
The start of an Induction Curve (under Kinetics, see 9.2) or a Light
Curve (see 9.3) is equivalent to the start of a New Record.
Previously defined areas of interest (AOI, see 9.1.2.2) are not erased
upon start of a New Record, such that several Records (e.g. Light
Curves and Induction Curves) can be measured for the same AOIs.
AOIs can be reset and newly defined at any time, in the Measure- as
well as in the View-mode.
8.1.3 Measure
With the help of the Measure-checkbox it is
possible to switch between the Measure- and the
View-modes. In the Measure-mode only the images recorded during
the last measurement (last Saturation Pulse) are displayed, whereas
in the View-mode all previously recorded data of a record can be
viewed. In the View-mode the functions located in the box at the left
apply. While actual measurements are possible in the Measure-mode
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only, the various types of illumination are not affected by switching
to the View-mode. In this way, it is possible to keep a sample in a
defined light state, while viewing previously recorded data. If the
user wants to stop illumination (e.g. in order to save battery power),
the various types of illumination (ML, AL and Clock) have to be
switched off manually. The Save-icon (see 8.2) is also accessible in
the Measure-mode. When it is clicked, the Measure-mode is
temporarily quit and the View-mode installed (see 8.2) for data
storage. After the data are saved, the Measure-mode is automatically
reinstalled, such that the running Record can be continued. Hence,
data can be saved successively in the course of a Record.
8.2
Functions applying to the View-mode
The View-mode is automatically installed when the Measuremode is quit (Measure checkbox). Data previously stored in the
Buffer-Memory can be viewed on the Image-, Kinetics-, Light
Curve- and Report-windows. For Kinetics and Light Curve at least
one Area Of Interest (AOI) must be defined (see 9.1.2.2). Previously
defined AOIs can be erased and new AOIs can be defined at any
time. The data in the Buffer-Memory are numbered according to the
time of measurement. A measurement is defined by application of a
Saturation Pulse. In the upper display line the number of
measurement with date and time is shown. Using the arrow bar
below, a particular measurement can be manually selected. To the
right of the display line the current number of measurement is
displayed in a separate box (also active in the Measuring-mode).
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Go
When Go is started, the images stored in BufferMemory are automatically displayed one after the
other at a rate determined by the Go Speed (under
Settings). After showing the last measurement, Go
automatically starts again with the first
measurement. Please note that the Yield-filter (see
9.5.12) slows down the image build-up of all
calculated parameters and, therefore, should be
switched off when high Go Speeds are chosen.
Save
The data transiently stored in Buffer-Memory can
be permanently saved on hard disk in form of a
PAM Image (PIM) file. Data saving is also
possible in the Measure-mode during the course of
a Record (see 8). Data are saved in the Datadirectory of the corresponding Imaging-PAM
version. Additionally to the Data file a commend
(.txt) file for experimental descriptions can be
edited and saved.
Open
Data stored in form of a PAM Image (PIM) file on
hard disk can be opened by loading into the BufferMemory. Then, if desired, also new AOIs may be
defined.
Show commend
Opens up the text file of the current PAM Image
file.
Export
Data stored in the Buffer-Memory can be exported
in the form of JEPG- or TIFF-files. A JEPG-file
serves for exporting one particular image, which
was selected for display on the Image-window and
is relatively small (c. 100 KB). On the other hand,
TIFF-files are rather large (c. 10 MB), as they
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essentially contain the same information as the
original PIM-files. Each TIFF file consists of a
series of images of the following parameters: Fo,
Fm, NIR, Red, F1, Fm'1, F2, Fm'2, F3, Fm'3 etc. In
principle, on the basis of these images, images of all
other fluorescence parameters as well as of PAR
Absorptivity can be derived (for formulas see
section 9.1.1). TIFF-images are monochrome
without false color coding. They are suited for
being used in conjunction with other image analysis
programs, like “ImageJ”.
8.3
Light controls
The Imaging-PAM employs the same LEDs for pulse-modulated
Measuring Light (ML), Actinic Light (AL), (Ext) and Saturation
Pulses.
ML
Checkbox for switching Measuring Light on/off at
a pulse frequency defined under Settings.
AL
Checkbox for switching Actinic Light on/off or to
start a period of actinic illumination, the duration of
which is defined under Settings. When AL is
switched on, ML frequency automatically is
switched to maximal setting 8.
Ext
Checkbox for switching an External Light Source
on/off intensity and width is defined under Settings.
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PAR
Display of light intensity (Photosynthetically
Active Radiation) in µmol quanta m-2 s-1, which is
corresponds to the intensity of Actinic Light as well
as (although to a much lesser extent) also by the
intensity and frequency of the Measuring Light, as
defined by the intrinsic PAR-List (under Options).
In the case of MAXI- and MINI-versions the
displayed values are calculated on the basis of
PAR-values measured at the given fixed distance
between LED-lamp and sample plane with the help
of a micro quantum sensor. With the
MICROSCOPY-version it also depends on the
choice of objective lens. With each measurement
(defined by a Saturation Pulse) the momentary
PAR-value is stored. It is also displayed in the
View-mode. The PAR-values for the 20 ALintensity settings, as well as for ML-frequency 8
(equivalent to AL0), are stored as default.par file in
the Data folder of each ImagingPam-version. It can
be viewed and/or modified under AL-List (LED
currents/PAR values see chapter 10.3). A
modification of the original values can e.g. become
necessary, when the optional Filter Plate
IMAG-MAX/F is used (see 3.2).
SAT-Pulse
Key for starting a single Saturation Pulse, which
defines a Measurement (i.e. determination of F and
Fm' as well as on-line calculation of the derived
fluorescence parameters), with the obtained data
being stored in the Buffer-Memory.
AL + Y
Key for starting a period of actinic illumination,
the length of which is defined under Settings (Act.
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Light Width) and at the end of which a Saturation
Pulse is applied. The AL + Y key is not active
when Act. Light Width is set to zero (indefinite).
Clock
When the Clock is switched on, the selected Clock
item is repeated with the set interval until manually
switched off again. The Clock interval can be set
between 5 s and 3600 s (1 hour). There is the choice
between four different Clock items: SAT. Pulse,
AL, AL + Y and Ft only. While the "SAT. Pulse"
and "AL + Y" Clocks involve the repetitive
application of Saturation Pulses and, hence,
correspond to the measurement of fluorescence
parameters, this is not the case for the "AL" Clock.
A particular case is the "Ft only" Clock, which
allows repetitive measurement of Ft without
application of a Saturation Pulse. In the absence of
actinic illumination, this allows to follow changes
in Fo or Fo'-images. In the case of the "AL" and
"AL + Y" Clocks, it should be made sure that the
Clock interval is longer than the Act. Light Width.
Using the SAT-Pulse clock not only single
Saturation Pulses but also sequences of defined
numbers of Saturation Pulses can be applied (see
9.5.1).
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9 IMAGINGWIN - Register Cards
9.1
Image-window
The major part of the Image-window is occupied by the actual
Image, at the bottom of which the false color code bar is located.
The standard false color code ranges from black via red, orange,
yellow, green, blue and violet to purple. These colors code for
numbers between 0 and 1. Hence, all measured or calculated
parameters are normalized to values between 0 and 1. The
correspondence between color and numerical value can be evaluated
with the help of a "Ruler" which can be installed above the false
color bar via Options in the menu. Instead of a false color bar also
the corresponding black-and-white bar (grey scale) can be installed
(via the B/W check box under Settings/Display). In the middle of the
Image, by default an area of interest (AOI) is defined in form of a
standard circle which is accompanied by a little red box displaying
the averaged value of the selected fluorescence parameter within this
AOI.
Below the Image-area the various parameters are listed, images
of which can be selected by clicking the corresponding radio buttons
(Select type of Image) (see 9.1.1). At the right hand side of the
Image-area a number of functional elements are located which serve
for image capture and analysis (see 9.1.2).
9.1.1 Different types of images
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Under Select type of Image one out of 18 different parameters
can be selected, the image of which is displayed on the Imagewindow. The meaning of the various parameters will be briefly
described in the following subsections.
9.1.1.1
Current fluorescence yield, Ft
The current fluorescence yield, Ft, is continuously monitored in
the Measure-mode (see 8), when the Measuring Light (ML) is
switched on. While images of Ft are not continuously stored in the
Buffer-Memory, at any time the current Ft image can be stored by
applying a Saturation Pulse. Then the current Ft-image is stored in
form of an F- or Fo-image. The latter applies, if the Saturation Pulse
is given in conjunction with an Fo, Fm-determination (see 8). It is
also possible to measure Ft-images without application of a
Saturation Pulse with the help of the "Ft only Clock" (see 8.3).
Kinetic changes of Ft can be recorded on the Kinetics-window in
conjunction with measurements of dark-light induction curves or
light response curves for selected areas of interest (AOI). In this case,
Ft-values are stored continuously, i.e. also between Saturation Pulses.
9.1.1.2
Dark fluorescence yield, Fo
The dark fluorescence yield, Fo, can be assessed after dark
adaptation using the Fo, Fm-key. After dark adaptation normally all
PS II reaction centers are open and maximal photochemical
quenching is observed. This does not necessarily mean that Fo is the
minimal fluorescence yield. Fluorescence yield can drop below the
Fo-level by strong non-photochemical quenching induced during
illumination. When an Fo measurement is triggered, the current Ft is
averaged for 3 s and the averaged value is denoted Fo. In
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averaging can be applied, which allows assessment of Fo at
substantially enhanced sensitivity (see 9.6.1). Fo determination is
essential for correct calculation of the quenching coefficient qP (see
9.1.1.16).
9.1.1.3
Fluorescence yield, F
The fluorescence yield, F, is assessed like all fluorescence
parameters (except for Ft) in conjunction with the application of a
Saturation Pulse. When a Saturation Pulse is triggered, the current Ft
is averaged for 3 s and the averaged value is denoted F. Like all
fluorescence parameters measured in conjunction with a Saturation
Pulse, F images are stored in the Buffer Memory.
9.1.1.4
Maximal fluorescence yield, Fm
Maximal fluorescence yield, Fm, can be assessed after dark
adaptation using the Fo, Fm-key. The Fm-value is assessed at the
plateau level reached during application of a Saturation Pulse.
During the Saturation Pulse the Measuring Light frequency
automatically is switched to the maximal setting. Assessment of Fm
involves averaging of 3 image recordings. In special applications,
when dealing with low signal levels (e.g. MAXI-version with algae
suspensions in multiwell plates or MICROSCOPY-version), a
"Special SP-Routine" can be applied, which allows assessment of Fm
at substantially enhanced sensitivity (see 9.6.1).
After dark adaptation normally the extent of energy-dependent
nonphotochemical quenching is minimal. Fo, Fm-determination at
the start of a New Record (see 8) is essential for correct calculation
of the quenching parameters qP, qN and NPQ. The Fm-image
measured at the start of a Record remains unchanged until a New
Record is started by a new Fo, Fm- determination. In this respect Fm
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differs from Fm', the images of which change with every Saturation
Pulse (see below).
9.1.1.5
Maximum fluorescence yield, Fm'
In illuminated samples, the maximum fluorescence yield, Fm', is
observed, which normally is lowered with respect to Fm by nonphotochemical quenching. Its value is assessed at the plateau level
reached during application of a Saturation Pulse. During the
Saturation Pulse the Measuring Light frequency automatically is
switched to the maximal setting. Assessment of Fm' involves
averaging of 3 image recordings. In special applications, when
dealing with low signal levels (e.g. MAXI-version with algae
suspensions in multiwell plates or MICROSCOPY-version), a
"Special SP-Routine" can be applied, which allows assessment of
Fm' at substantially enhanced sensitivity (see 9.6.1).
A given sample can show an infinity of different Fm'-images,
depending on the state of illumination at the very moment when the
Saturation Pulse is applied. On the other hand, the same sample is
characterized by unique Fo- and Fm-images, which are determined
with a dark adapted sample (see 9.1.1.4 and 9.1.1.2).
9.1.1.6
Maximal PS II quantum yield, Fv/Fm
Maximal PS II quantum yield, Fv/Fm, is determined after darkadaptation. It is calculated according to the equation:
Fv/Fm = (Fm - Fo)/Fm
After dark adaptation normally all PS II reaction centers are open
(F = Fo) and non-photochemical energy dissipation is minimal (qN =
NPQ = 0) and maximal fluorescence yield, Fm, is reached during a
Saturation Pulse. In this state the fluorescence increase induced by a
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Saturation Pulse (variable fluorescence, Fv) as well as the PS II
quantum yield (F/Fm = Fv/Fm) are maximal. The Fv/Fm image is
measured in conjunction with an Fo, Fm-determination. It remains
unchanged until the next Fo, Fm-determination. In this respect, the
Fv/Fm image differs from the Y(II) image which changes with every
Saturation Pulse (see below).
The contrast between the photosynthetically active object and the
background matrix is enhanced, by the definition: Fv/Fm = 0 if
Fm <0.048. All pixel for which this limit is not reached, are
displayed in black. In this way, unavoidable noise associated with the
Fm-determination can be suppressed. The resulting "noise mask" is
saved for a given Record and also applied to Y(II), Y(NPQ) and
Y(NO) images. Please note that this approach requires that the
sample does not move during a given Record. If sample movement
cannot be avoided, quenching analysis is not possible and
measurements should be carried out without Fo, Fm-determination.
9.1.1.7
Effective PS II quantum yield, Y(II)
The effective PS II quantum yield is calculated according to
Genty et al. (1989) by the formula:
Y(II) = (Fm'-F)/Fm'
As this fluorescence parameter is derived from a ratio of
fluorescence intensities, any inhomogeneities of fluorescence
excitation intensity or chlorophyll concentration will disappear and
any remaining inhomogeneities can be interpreted in terms of
differences in activity.
A given sample can show an infinity of different Y(II)-images,
depending on the state of illumination at the very moment when the
Saturation Pulse is applied. A unique state is given after dark
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adaptation when the effective PS II quantum yield is maximal (see
9.1.1.6).
Y(II) measurements normally are preceded by an Fo, Fmmeasurement. In this case the contrast between the
photosynthetically active object and the background matrix is
enhanced, by the definition: Fv/Fm = 0 if Fm<0.048. All pixel for
which this limit is not reached, are displayed in black in Fv/Fm as
well as in Y(II) images ("noise mask", see 9.1.1.6).
In principle, a quantum yield may vary between 0 and 1. If, for
example Y(II) = 0.5, this means that one half of the absorbed quanta
are converted into chemically fixed energy by the photochemical
charge separation at PS II reaction centers. The other half of the
quanta is dissipated into heat and fluorescence. The sum of all
quantum yields always amounts to 1. Based on the work of Kramer
et al. (2004) Photosynthesis Research 79: 209-218 two other types of
quantum yield can be defined, Y(NPQ) and Y(NO), which represent
the nonregulated and regulated energy dissipation at PS II centers,
respectively (see 9.1.1.8 and 9.1.1.9), adding up to unity with the
photochemical quantum yield:
Y(II) + Y(NPQ) + Y(NO) = 1
9.1.1.8
Quantum yield of regulated energy dissipation, Y(NPQ)
The quantum yield of regulated energy dissipation in PS II,
Y(NPQ) can be calculated according to Kramer et al. (2004) by the
equation:
Y(NPQ) = 1 - Y(II) - 1/(NPQ+1+qL(Fm/Fo-1))
For the validity of this equation it is essential that the PS II
pigments of the investigated sample are organized according to the
"Lake model" (Stern-Volmer approach), which may be assumed for
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most higher plant leaves. The NPQ parameter is a measure of
nonphotochemical fluorescence quenching (see 9.1.1.13), reflecting
down-regulation of PS II as a protective mechanism against excess
light intensity. The qL parameter is a measure of the fraction of open
PSII centers in the "Lake model" (see 9.1.1.17).
A high Y(NPQ) value on one hand indicates that the photon flux
density is excessive and on the other hand shows that the sample has
retained the physiological means to protect itself by regulation, i.e.
the dissipation of excessive excitation energy into harmless heat.
Without such dissipation there would be formation of singlet oxygen
and reactive radicals, which cause irreversible damage.
In Y(NPQ) images all pixel are set to 0 (black) for which
Fv/Fm = 0 ("noise mask", see 9.1.1.6).
9.1.1.9
Quantum yield of nonregulated energy dissipation,
Y(NO)
The quantum yield of nonregulated energy dissipation in PS II,
Y(NO) can be calculated according to Kramer et al. (2004) by the
equation:
Y(NO) = 1/(NPQ+1+qL(Fm/Fo-1))
For the validity of this equation it is essential that the PS II
pigments of the investigated sample are organized according to the
"lake model" (Stern-Volmer approach), which may be assumed for
most higher plant leaves. The NPQ parameter is a measure of
nonphotochemical fluorescence quenching (see 9.1.1.13), reflecting
down-regulation of PS II as a protective mechanism against excess
light intensity. The qL parameter is a measure of the fraction of open
PSII centers in the "lake model" (see 9.1.1.17).
A high Y(NO) value indicates that both photochemical energy
conversion and protective regulatory mechanisms are inefficient.
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Therefore it is indicative of the plant having serious problems to
cope with the incident radiation. Either it is already damaged or it
will be photodamaged upon further irradiation. Extremely high
values of Y(NO) e.g. can be induced by PS II herbicides, which not
only block PS II reaction centers, but also prevent the build up of a
transthylakoidal proton gradient. The latter is an important
prerequisite for energy-dependent non-photochemical quenching.
In Y(NO) images all pixel are set to 1 (purple) for which
Fv/Fm=0 ("noise mask", see 5.4.1.6).
9.1.1.10 Absorptivity, Abs.
Measurement of the Absorptivity parameter requires a special set
of Red and NIR LEDs that are integrated in the LED-arrays of the
MAXI- and MINI-Heads in the blue measuring light version. The
red measuring light versions as well as the MICROSCOPY-version
of the Imaging-PAM M-series do not support this function.
The Absorptivity (Abs.) image is a measure of the fraction of the
incident Red-light which is absorbed by the leaf sample. It is
recorded with the help of the "Measure Abs." function (see 9.1.2.1).
When the Measure Abs. key is pressed, automatically the sample is
first illuminated with Red and then with NIR light and the R- and
NIR-images are recorded. The apparent Absorptivity is calculated
pixel by pixel from the R- and NIR-images using the formula.
Abs. = 1 - R/NIR.
This measurement is based on previous calibration of the
instrument, which involves appropriate adjustment of the R-intensity
with respect to the NIR-intensity. The instrument is properly
calibrated, when the R- and NIR-images of a white piece of paper (or
white flower petal) show similar brightness values. Then R/NIR is
close to unity and Abs. = 1 - R/NIR is close to zero. Any pigment
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that absorbs Red more than NIR light, i.e. generally all
photosynthetically active pigments, will decrease R with respect to
NIR, thus decreasing R/NIR and increasing the derived Abs.-value.
On the other hand, pigments that absorb Red and NIR light similarly,
as e.g. necrotic spots, will not cause R/NIR to deviate substantially
from unity and thus Abs. will remain close to zero. Notably, a white
and a black piece of paper give similar Abs.-images with pixel-values
close to zero. This illustrates that the Abs. parameter does not assess
general Absorptivity of a sample for visible light, as sensed by the
human eye, but rather specifically the Absorptivity of
photosynthetically active light. Therefore, the Abs. parameter as
determined by the IMAGING-PAM may be considered a close
estimate of PAR-Absorptivity. This approach is based on the
empirical fact that pigments that contribute to the absorption of PAR
do not show significant absorption bands in the near-infrared (NIR)
spectral region. On the other hands, pigments that absorb NIR are
likely to also absorb Red light.
For technical reasons, the measurement of PAR-Absorptivity is
carried out using red light (660 nm), whereas blue light is employed
for actinic illumination (450 nm). This approach may be justified by
the fact that the same pigments (mainly chlorophyll a and b) are
responsible for absorption at 660 as well as at 450 nm. PARAbsorptivity complements the information provided by the Yieldimage on the lateral distribution of effective PS II quantum yield.
The incident PAR is known as it is defined by the
MAXI-IMAGING-PAM, provided that illumination by ambient light
can be neglected. With knowledge of Yield, PAR and Abs. the
apparent rate of photosynthetic electron transport rate (PS-parameter)
can be estimated (see 9.1.1.11).
The absorbed PAR may be overestimated when leaves contain
PAR-absorbing pigments which do not transfer the absorbed energy
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to the photosynthetic reaction centers. A special case is given when
leaves are red colored, e.g. due to anthocyanin accumulation in the
vacuoles of the epidermis. On one hand, this will cause a low Abs.value, as a large fraction of the Red will be remitted. On the other
hand, also the amount of blue light penetrating to the mesophyll will
be lowered, leading to correspondingly low values of absorbed PAR
and fluorescence yield.
It should be noted that the Abs.-value does not necessarily
correlate with chlorophyll content per leaf area (as commonly
determined by extraction). For example, if the leaf surface is covered
by reflecting hairs (pubescence) this will strongly affect PARAbsorptivity, thus lowering the measured Abs.-values, without
affecting leaf chlorophyll content. The same leaf normally shows
considerably lower Abs.-values at the lower side compared to the
upper side.
9.1.1.11 Apparent rate of photosynthesis, PS/50
An apparent rate of photosynthesis (PS) can be calculated on the
basis of the measured effective PS II quantum yield, the incident
photon flux density (PAR) and the PAR-Absorptivity (Abs.):
PS = 0.5 x Y(II) x PAR x Abs. µequivalents m-2 s-1
The incident PAR is known for the defined distance between
LED-Ring-Array and sample plane. The PAR-Absorptivity
previously has to be measured using the Measure Abs. routine (see
9.1.1.10). It is assumed that 50 % of the absorbed PAR is distributed
to PS II. As the PS calculated in this way can reach values around 50,
the parameter PS/50 is displayed in order to depict the apparent rate
of photosynthesis with the help of the false color code for values
ranging between 0 and 1.
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The PS-parameter measured with the IMAGING-PAM in most
respects closely corresponds to the ETR-parameter previously
defined for measurements of the apparent rate of photosynthetic
electron transport:
ETR = 0.5 x Yield x PAR x 0.84 µequivalents m-2 s-1
This commonly used definition assumes a PAR-Absorptivity of
0.84, i.e. that 84 % of the incident photons of photosynthetically
active radiation is absorbed by the leaf. This value may be
considered typical for a "standard green leaf". In reality, however,
PAR-Absorptivity may vary considerably, e.g. between upper and
lower leaf side, as well as between different species and leaves at
different developmental stages. The latter is particularly evident
during senescence. Even the same leaf may show considerable lateral
heterogeneity of PAR-Absorptivity, in particular caused by virus
infection and other plant diseases that disturb pigment synthesis.
9.1.1.12 NIR light remission, NIR
NIR light remission, NIR, is measured in conjunction with the
function "Measure Abs." (see 9.1.2.1). It provides a measure for the
remission of light that is not absorbed by photosynthetically active
pigments. For this purpose 780 nm light is applied. The remission
includes both reflection and backscattering of the 780 nm light
received by the CCD detector. The reflection mainly occurs at the
leaf surface, i.e. before the incident light has reached the cell layers
containing photosynthetic pigments, and therefore should be similar
for 660 nm and 780 nm light. On the other hand, the backscattering
is very different for 660 nm and 780 nm light, as most of the 660 nm
light in contrast to the 780 nm light is absorbed by the photosynthetic
pigments. In this respect, backscattering is similar to transmission,
which in a leaf is strongly influenced by light scattering that causes a
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considerable increase of the path length of the measuring light within
the leaf.
The intensity of the NIR-measuring light can be adjusted under
Live Video (see 9.1.2.3). It should be made sure that there is no
overload, particularly under conditions when the non-modulated
background signal is relatively large (due to ambient light). While
the non-modulated background signal does not contribute to the
intensity values of the NIR-image, it does add to an overload of the
overall signal. Once the instrument is calibrated for measurements of
PAR-Absorptivity (see 9.1.1.10), changes in NIR-measuring light
intensity should be avoided, as otherwise the Red-measuring light
intensity would have to be readjusted.
Please note that the NIR-image is not corrected for heterogeneity
of intensity distribution, which in this case mostly consists in a
decrease of brightness on the fringes of the image caused by the
vignetting effect of the objective lens. As essentially the same effect
is observed in the R-image, it does not affect the ratio R/NIR and the
calculated Absorptivity parameter Abs. = 1 - R/NIR.
NIR-images cannot be measured with the MICROSCOPYversions of the Imaging-PAM.
9.1.1.13 Nonphotochemical quenching, NPQ/4
The NPQ parameter provides a measure of nonphotochemical
quenching which in contrast to qN does not require knowledge of Fo'
(see 9.1.1.2). It is defined according to the equation:
NPQ = (Fm-Fm')/Fm'
In contrast to the quenching coefficient qN, the parameter NPQ
can reach values higher than 1. In practice, NPQ rarely exceeds 4.
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Hence, images of NPQ/4 are displayed, with values ranging between
0 and 1, that can be displayed using the standard false color code.
The definition of NPQ implies a matrix model of the antenna
pigments (Stern-Volmer quenching). With NPQ that part of
nonphotochemical quenching is emphasized which reflects heatdissipation of excitation energy in the antenna system. NPQ has been
shown to be a good indicator for "excess light energy" which in
leaves is primarily dissipated via zeaxanthin (xanthophyll cycle) in
the presence of a transthylakoidal pH. On the other hand, NPQ is
relatively insensitive to that part of nonphotochemical quenching
which is associated with qN-values up to 0.4, reflecting thylakoid
membrane energization.
Assessment of NPQ requires previous determination of Fm with
the same sample after dark adaptation, i.e. under conditions when per
definition NPQ = 0.
9.1.1.14 Red light remission, R
Red light remission, R, is measured in conjunction with the
function "Measure Abs." (see 9.1.2.1). It is an inverse measure of
the absorption of photosynthetically active radiation (PAR) by the
investigated sample. The 660 nm light emitted by the red LEDs in
the LED-Ring-Array is strongly absorbed by chlorophyll. Hence,
only a small fraction of the incident red light is remitted (i.e.
reflected and backscattered) from the sample. The same is true for
the 450 nm light serving for fluorescence excitation and actinic
illumination. However, in contrast to 660 nm, 450 nm cannot
penetrate to the CCD-detector, which is protected by a red filter. The
intensity of the red light is adjusted such that a highly scattering
white sample (white piece of paper or white flower petal) gives the
same signal as with the near-infrared (780 nm) reference light. The
780 nm light is emitted by LEDs adjacent to the 660 nm LEDs in the
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LED-Ring-Array (3.2). Both types of LEDs emit rather diffuse light,
thus giving very homogenous illumination of the sample. In contrast
to the 660 nm light, the 780 nm light is not absorbed by leaves, as
chlorophyll absorption does not extend into this wavelength range.
Please note that the R-image is not corrected for heterogeneity of
intensity distribution, which in this case mostly consists in a decrease
of brightness at the fringes of the image caused by the vignetting
effect of the objective lens. As essentially the same effect is observed
in the NIR-image, it does not affect the ratio R/NIR and the derived
Absorptivity parameter Abs. = 1 - R/NIR.
Red-images cannot be measured with the MICROSCOPYversions of the Imaging-PAM.
9.1.1.15 Coefficient of nonphotochemical quenching, qN
The coefficient of non-photochemical quenching, qN, is defined
by the equation:
qN = (Fm-Fm')/(Fm-Fo')
qN can vary between 0 (defined for dark adapted state) to 1 (all
variable fluorescence quenched). The above definition takes into
account that not only variable fluorescence (induced upon reaction
center closure), but also the dark-level fluorescence (all centers open)
can be quenched non-photochemically, primarily by increased heat
dissipation induced during illumination. For correct determination of
Fo', it would be necessary to switch off the actinic light and to
quickly reoxidize the PS II acceptor side with the help of far-red
light, before non-photochemical quenching can relax. This approach,
however, is not feasible with the IMAGING-PAM, as the far-red
light would penetrate to the CCD-detector and cause serious
disturbance of fluorescence imaging. Therefore, instead of measuring
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Fo', this parameter is estimated using the approximation of
Oxborough and Baker (1997):
Fo' = Fo/ (Fv/Fm + Fo/Fm')
This approximation relies on the assumption that the same
mechanism that causes quenching of Fm' with respect to Fm is also
responsible for Fo-quenching. The quenching coefficient qN is quite
sensitive to changes in the energy status of the chloroplasts (energydependent quenching). Such changes are readily induced by various
environmental stress factors causing stomatal closure, switching
from CO2-dependent to O2-dependent electron flow and downregulation of the rate of energy conversion in PS II. Hence, qN is an
indicator of stress induced limitations and, actually, has proven to be
the most sensitive parameter for early detection of such limitations
by fluorescence imaging.
Assessment of qN requires previous Fo, Fm-determination with
the same sample after dark adaptation, i.e. when qN = 0 per
definition.
9.1.1.16 Coefficient of photochemical quenching, qP
The coefficient of photochemical quenching, qP, is a measure of
the overall "openness”. Red-images cannot be measured with the
MICROSCOPY-versions of the Imaging-PAM. They vary between 0
and 1. Calculation of qP requires knowledge of the fluorescence
parameter Fo' (minimal fluorescence yield of illuminated sample,
which is lowered with respect to Fo by non-photochemical
quenching):
qP = (Fm'-F)/(Fm'-Fo')
Correct Fo'-determination requires application of far-red light,
which would disturb the fluorescence imaging. However, as the same
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mechanism causing Fo-quenching is also responsible for quenching
of Fm' with respect to Fm, it is possible to estimate Fo' from Fm'
measurements (Oxborough and Baker 1997):
Fo' = Fo/ (Fv/Fm + Fo/Fm')
Assessment of qP requires previous Fo-Fm determination with
the same sample after dark adaptation, i.e. when qP = 1 per
definition.
While the definition of qP is based on the "puddle model" of PS
II, the antenna pigment organization in leaves is more realistically
described by the "lake model". This means that the antenna of
individual PS II reaction centers are connected, so that the excitation
energy can be transferred with high probability from closed reaction
centers to neighboring open centers. Therefore, the fraction of open
PS II centers is overestimated by qP. The fraction of open PS II
centers estimated on the basis of the "lake model" is described by the
quenching coefficient qL (see below).
9.1.1.17 Coefficient of photochemical quenching, qL
The coefficient of photochemical quenching, qL, is a measure of
the fraction of open PS II reaction centers, which can vary between 0
and 1. Its definition is based on the "lake model" of PS II antenna
pigment organization. Calculation of qL requires previous
determination of the fluorescence parameter Fo' (minimal
fluorescence yield of illuminated sample, which is lowered with
respect to Fo by nonphotochemical quenching):
qL = (Fm'-F)/(Fm'-Fo') x Fo'/F = qP x Fo'/F
Correct Fo'-determination requires application of far-red light,
which would disturb the fluorescence imaging. However, as the same
mechanism causing Fo-quenching is also responsible for quenching
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of Fm' with respect to Fm, it is possible to estimate Fo' from Fm'
measurements (Oxborough and Baker 1997):
Fo' = Fo/ (Fv/Fm + Fo/Fm')
Assessment of qL requires previous Fo-Fm determination with
the same sample after dark adaptation, i.e. when qL = 1 per
definition.
When during illumination nonphotochemical quenching is
generated, generally Fo'<F and, therefore, also qL<qP. The difference
between these two coefficients of photochemical quenching increases
with the connectivity between PS II reaction centers.
9.1.1.18 Inhibition, Inh.
The Inhibition (Inh.) parameter describes the inhibition of PS II
quantum yield, Fv/Fm or Y(II), relative to a control reference AOI,
the number of which can be selected under Settings / Inh. Ref. AOI
(see 9.5.11). After start of the program AOI #1 is the control
reference for calculation of the Inh. image according to the equation:
Inh. = (Ycontrol - Ysample) / Ycontrol
This parameter is particularly important for assessment of
phytotoxicity with multiwell plates using the MAXI-version. In this
application wells are filled with algae suspensions and the inhibitory
effect of phytotoxicant addition relative to a control sample is
assessed. In this case AOI #1 is defined for the well of the control
sample, which always displays the highest Y(II) value.
Images of Inh. can also be informative in applications with other
objects, like leaves, using all versions of the Imaging-PAM. In
phytopathological studies, for example, the inhibition of an infected
area relative to a control area (defined as AOI #1) can be depicted.
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Like all other imaged parameters, the Inh. ranges from 0 (black)
to 1 (purple). In order to assure a good contrast between sample and
background, in the case of Inh. images the latter is white instead of
the usual black.
9.1.2 Image capture and analysis
The functional elements for image capture and analysis are
located at the right hand side of the Image-window. The standard
Image-window is shown when Capture is selected. The measured
image of a particular parameter can be modified for emphasizing
certain features when Analysis is selected. The various functional
elements are described in the following sub-sections.
9.1.2.1
Measure Abs.
This function requires a special set of Red and NIR LEDs that
are integrated in the LED arrays of the MAXI- and MINI-Heads in
the blue measuring light version. The MICROSCOPY-versions of
the Imaging-PAM M-series does not support this function.
Using the Measure Abs. key an automatic routine for the
measurement of a PAR-Absorptivity (Abs.) image can be started
(see also 9.1.1.10). This measurement involves consequent
illumination of the sample with Red and NIR light, resulting in the
capture of Red and NIR remission images (see 9.1.1.14 and
9.1.1.12), from which the Abs.-image is calculated pixel by pixel
using the formula:
Abs. = 1 - R/NIR.
As the Abs., Red and NIR images are erased upon start of a New
Record, it is recommended to carry out an Abs. measurement
routinely after start of a New Record (see 8).
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The Measure Abs. routine can give meaningful results only, if the
relative intensities of the NIR and Red measuring light are properly
adjusted (see also 9.1.1.10). Appropriate settings of NIR and Red
intensity, as well as of Red Gain, were determined at the factory for
each individual IMAGING-PAM and are documented on a sticker
fixed to the corresponding LED-Array. These settings are
preinstalled on the Notebook PC IMAG-PC, if this is purchased
together with the instrument. They are listed in the INI-file within
the ImagingPAM directory. Correct calibration can be ascertained
by the user by measuring Red and NIR images of a white piece of
paper. While it appears unlikely that the settings of NIR and Red
intensities change with time, small corrections of Red Gain may be
required after some time, due to ageing of the LEDs. For this
purpose, the Red Gain is accessible under Settings/Absorptivity (see
also 9.5.3). No corrections may be necessary, if the Red and NIR
values do not differ by more than 5 %. If necessary, also the values
of Red and NIR intensities can be corrected. These parameters are
accessible under Settings/Absorptivity.
9.1.2.2
Area of Interest, AOI
The displayed image is composed of 640 x 480 (i.e. 307200)
pixels. Each pixel captures specific fluorescence information, such
that in principle e.g. 307200 Light Response Curves could be
recorded. In practice, however, it is necessary to
reduce this vast amount of information. For this
purpose, special Areas of Interest, AOI, can be
defined. All pixel values contained in an AOI are
averaged and the averaged value is shown in a
little box adjacent to a particular AOI. In this way, the pixel noise is
considerably reduced.
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The definition of at least one AOI is
required for recordings of Induction Curves
(see 9.2) and Light Curves (see 9.3). After
start of the program a standard AOI circle is
installed by default in the center of the image
area. This AOI can be removed with the help of the Reset-button.
New AOIs can be installed with the help of the Add-button. After
clicking "Add", a standard circle can be moved with the help of the
mouse cursor to the desired position. With the help of the + key the
circle diameter can be increased. With the - key it can be decreased.
The AOI position and size are confirmed by mouse click (left or
right). The AOI-size will remain unchanged when further AOIs are
added, unless it is modified using +/-. The last added AOI can be
removed via Edit/Undo (Menu). All AOIs can be removed by Reset.
Any particular single AOI can be deleted via Delete. After clicking
the Delete button a Delete-hand appears that can be moved to a
particular AOI using the mouse. Please note that the pointing finger
has to cross the border of the AOI. When the Show-checkbox is
deactivated, the AOIs disappear, but can be recalled at any time by
checking the box. The status of the Filled-checkbox determines
whether or not the area is filled with the color corresponding to the
averaged parameter value. When "Filled" is activated, any
heterogeneity or structure within the AOI disappears. Only when
"Filled" is inactivated it is possible to see a small AOI covered by a
larger AOI.
With the help of the Type-button different AOI types can be
selected. Besides a Circle also a Rectangle or Polygon can be
chosen. The minimal AOI size is one pixel with a rectangular and a
diameter of 10 pixels with a circular AOI.
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The AOI Type has to be selected before clicking "Add"; after
clicking "Add" the Type-selection is not accessible.
For definition of the size and position of a Rectangle AOI, after
clicking "Add" the mouse cursor is moved to one of the envisaged
corners, which is fixed by a mouse click (left or right). Then the
desired shape and size of the Rectangle can be arranged by mouse
movement. The final state is fixed by another mouse click.
For definition of the size and position of a Polygon AOI, after
clicking "Add" the mouse is moved to one of the envisaged corners,
which is fixed by a mouse click (left or right). Then the mouse cursor
is moved to the next corner, which is fixed by mouse click and so on.
The last corner is marked by a double-click.
Up to 100 AOI can be defined. A special routine is provided for
definition of an AOI-array, as e.g. required for assessment of
samples in multiwell plates. This routine is accessible under
Options/Define AOI array geometry/Create AOI array (see chapter
10.2)
9.1.2.3
Select: Fluorescence or Live Video
After start of the program the measuring
system is in the Fluorescence Mode. This
means that the fluorescence yield is assessed
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by the pulse modulated blue measuring light. In this way the
momentary Ft image is continuously captured. A rather low pulse
frequency is applied in order to avoid an actinic effect of the
measuring light. Consequently changes of fluorescence images are
strongly damped and it is difficult to follow the fluorescence image
while moving the sample or trying to focus the image.
When the Live Video Mode is selected, the blue fluorescence
measuring light is switched off and instead the NIR measuring light
is switched on, which is neither seen by human eyes nor sensed by
the plant. However, the NIR is detected by the CCD camera and thus
can serve for imaging the leaf (monochrome) using a relatively
high measuring pulse frequency, without causing preillumination of
the photosynthetic apparatus. Hence the Live Video Mode is useful
for positioning the leaf sample in the field of view and for focusing
the image.
When Live Video is activated, the Live Video window appears,
on which the NIR intensity can be adjusted.
When the Overload box on the Live Video window is activated,
this indicates that the overall amount of light seen by the CCD
camera is too high and, hence, the measured NIR image is likely to
be disturbed. The overall signal consists of the ambient background
light (e.g. from daylight) and the remitted NIR light. Hence, in the
case of Overload, either the incidence of ambient light should be
reduced (recommended) or the NIR intensity decreased.
Live Video images can be also obtained with continuous light of
any external light source, if this light can pass the filters in front of
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the CCD camera. This feature is particularly important in
Microscopy-applications using the standard through-light
condenser-illuminator of the microscope. In this way samples can
be readily centered and focused using the PC monitor screen.
In order to quit the Live Video Mode and to return to the
Fluorescence Mode click the Close button on the right hand side or
the Exit button in the upper right corner of the NIR intensity
adjustment window.
9.1.2.4
Zoom
Standard display of images (Zoom out)
involves all 640 x 480 (i.e. 307200) pixels.
While without Zoom single pixels cannot be
seen, they become more and more visible,
when the Zoom factor is increased. The
standard Zoom factor 2 is applied when
Zoom in is clicked. Then the 1/4 area in the
center is displayed. At that magnification the individual pixels are
just visible. The Zoom factor can be defined by the user after
clicking the Define Zoom button, which is possible only in the
Zoom out position. After Define Zoom is clicked, the mouse cursor
arrow tip has to be moved to one of the corners of the envisaged
Zoom image. This corner is fixed by a mouse click (left or right).
Then the desired size of the Zoom image can be adjusted by
movement of the diagonal arrow into the diagonally opposite corner
of the image rectangle. The final state is fixed by another mouse
click. In order to change the Zoom factor, always first Zoom out
must be selected, before Define Zoom can be clicked again. The
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Reset button serves for reset of the standard Zoom with Zoom
factor 2 (display of central quarter).
9.1.2.5
Cursor
The Cursor box shows the numerical value
of the selected parameter at the cursor position
which can be changed by mouse movement.
When the cursor enters an AOI the cursor box
will show the same averaged value as shown in
the box close to the AOI, provided "Filled" is
active (see 9.1.2.2).
The boxes above the Cursor box show the parameter values
applying to the whole image:
Min. : the minimal value of all pixels
Max. : the maximal value of all pixels
Mean : the mean value of all pixels
9.1.2.6
Analysis
The Analysis function normally is used in conjunction with
Expanded Color display (see 9.5.8). When Analysis instead of
Capture is selected, the color scale of a displayed image can be
modified. Using the Low and High scroll boxes, the Low-High
limits of the color scale can be defined. The numbers correspond to
the scale of pixel values ranging from 0.000 to 1.000. In the image
displayed under Analysis all pixels with values within the Low-High
limits are displayed in red color, while the rest of the pixels is
displayed in black-and-white (standard grey scale). The Low-High
limits defined under Analysis are effective for the Expanded Color
display under Capture. The closer the low and high limits are with
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respect to each other, the more expanded is the color display. In this
way, small differences in pixel values can be emphasized by
enhanced color differences, thus increasing the contrast.
Using the false color scale maximal contrast is obtained in the range
of very low values (from yellow to red and black). Therefore, in
order to obtain maximal contrast in the display of a selective
lowering of a fluorescence parameter in a particular region of a leaf,
the "normal" range of pixel values should be shifted to yellow while
the lowered range of pixel values should be shifted to red-black. This
can be achieved by suitable shifting of the Low and High limits
under Analysis. Under Settings the Expanded Color display has to
be selected and the image viewed under Capture. An example is
given in Fig. 55 shows a Y(II)-image in normal Color display of a
leaf in which the veins display lowered Yield-values. In Fig. 56 the
Analysis-image and the settings of the Low-High limits are
displayed. Fig. 57 shows the same image with Expanded Color
display under Capture.
Fig. 55: Normal color display (under capture)
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Fig. 56: Display under Analysis with particular settings of Low-High limits
Fig. 57: Expanded Color display (under Capture)
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Kinetics window
On the Kinetics window the changes of fluorescence parameters
are plotted versus time. Following every Fo, Fm-determination, i.e.
after start of a new Record, all measurements are saved in the Buffer
Memory, as protocolled in the lower left corner of the ImagingWin
user surface (see 8.2) and in more detail in the Report file (see 9.4).
The same information can be displayed in form of kinetic curves on
the Kinetics window. Registration of the kinetic data also occurs in
the background, i.e. when the Kinetics window is not active. Display
of data in the Kinetics window requires that at least one AOI is
selected. If this is not the case, there is a corresponding warning.
Fig. 58: Kinetics window with display of standard Induction Curve
A typical Record of a standard dark-light Induction Curve is
displayed in Fig. 58. For the sake of clarity only part of the available
fluorescence parameters is displayed (Fm', Y(II), qN and Ft) and
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only one AOI is selected. The Ft-parameter (momentary
fluorescence yield at any time, t), differs from all other parameters in
that it is measured continuously, i.e. not only in conjunction with
Saturation Pulses. However, it should be noted that Ft is recorded
exclusively for the selected AOIs. Hence, in contrast to all other
parameters, it is not possible to display Ft images for AOIs defined
after the recording. Also in contrast to all other parameters Ft is not
recorded in the background after Start of a New Record (Fo, Fmdetermination).
For the sake of a uniform ordinate scale reaching from 0 to 1, Ft
is referenced to the Fm-value, i.e. the ratio Ft/Fm is plotted versus
time. Hence, before a Kinetics curve can be recorded, Fm must be
determined, which is done automatically upon start of a recording via
an Fo, Fm determination, unless the user wants to keep previously
determined Fo, Fm values. With the definition of Fo, Fm a new
Record is started and the buffer memory with the previously
recorded data is erased. Therefore, the user is reminded to save the
data before the Fo, Fm-measurement is initiated or the current
Fo, Fm can be confirmed:
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For example, it can be advantageous to keep the previously
recorded Fo,Fm, when an Induction Curve is measured after a
defined preillumination. In this case, the Fo,Fm can be measured
before the preillumination and Fo,Fm determination preceding the
recording of the Induction Curve can be omitted. Then Ftnormalization as well as calculation of quenching parameters and of
Fv/Fm will be based on the previously determined Fo and Fm values.
When the AOI button is
pressed, the AOI window is
opened, which shows in its lower
part a list of all AOIs previously
defined on the Image-window. By
clicking a particular number, the
corresponding AOI can be
selected for data display in the
Kinetics window. The data of
several or all AOIs may be
superimposed. In the upper part of
the AOI window the data point
symbols of the selected AOI
number are shown. Examples are
given for one out of 4 selected AOIs and 8 out of 8
selected AOIs being active for display, respectively.
The time-dependent changes of eleven different
fluorescence parameters may be displayed in the
Kinetics window. For display the corresponding
check box has to be marked. As pointed out above,
the Ft parameter can be displayed for AOIs only
which were defined before start of the Record.
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One out of three different types of
Kinetic recordings can be selected
(Induction Curve, Manual recording
and Induction + Recovery). After Start
of a recording, it can be terminated via
the Stop-button. Next refers to the
remaining time until the next measurement takes place (i.e.
application of next Saturation Pulse).
Ind. Curve: An Induction Curve is a preprogrammed dark-light
induction curve (Kautsky effect), the parameters of which can be
defined by the user (under Settings/Act. Light/Slow Induction).
After Start of an Induction Curve, normally first an Fo, Fm
determination is carried out (unless the user prefers to keep the
previously determined Fo,Fm values, see above). Actinic
illumination is started after the Delay-time at an intensity defined by
Act. Light Int. Saturation Pulses for quenching analysis are given
repetitively at defined Clock-intervals. The length of the recording is
defined by the Duration-parameter. An Induction Curve is
terminated automatically at the end of the preprogrammed Durationperiod. It can be terminated earlier with the help of the Stop-button,
however not before the Delay-time is passed.
Manual:
The Manual recording corresponds to a chart
recording. When the Ft checkbox is activated, for all AOIs the time
courses of the averaged Ft pixel values are displayed. After Start of a
Manual registration, normally first an Fo, Fm determination is
carried out. The user may also decide to keep the previously
determined Fo, Fm values. It is up to the user when to start actinic
illumination, to apply a single Saturation Pulse or repetitive
Saturation Pulses using the Clock (at lower right corner of the
screen). A Manual registration is terminated by the Stop-button.
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Ind.+Rec.:
An Induction Curve + Recovery is a preprogrammed
dark-light induction curve (Kautsky effect) followed by a light-dark
induction curve which provides information on the dark-recovery of
fluorescence parameters after a period of illumination, the parameters
of which can be defined by the user. The Duration-parameter
(defined under Settings/Slow Induction) refers to the period of
illumination (just like in the case of a normal Induction Curve). In
the time period following termination of actinic illumination 16
Saturation Pulses are applied with the time between Saturation
Pulses being exponentially increasing. In this way the rapid recovery
kinetics can be recorded briefly after light-off and during registration
of the slow recovery kinetics the actinic effect of the Saturation
Pulses is minimized. Fig. 59 shows a typical recording of an
Induction Curve+Recovery. In this example the Duration-parameter
was set to 600 sec.
Fig. 59: Typical Induction Curve+Recovery
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When the Autoscale icon is clicked the time scale
automatically is changed such that the data fill the Kinetics
screen.
The X-Y boxes
show the coordinates of
the cursor position, with
X corresponding to the time (sec) and Y corresponding to the pixel
value of the selected fluorescence parameter.
When the cursor is moved on
top of a data point, for a period of
10 sec the number of the
measurement and the values of the selected parameters are shown.
An event marker
can be set in form of a
vertical red line and a
corresponding
event
text may be entered.
For this purpose, the
cursor has to be moved
to the time of the event and the right mouse has to be clicked. Then
the Set Marker box is opened, into which the event text may be
entered. After confirmation by OK, the red vertical line is installed
and whenever the cursor comes close to this line the event text is
displayed for 10 sec. The event marker is saved in the pim-file.
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Light Curve window
Fig. 60: Light Curve window with display of typical Light Curve of the
ETR-parameter
Recording of a Light Curve consists of a number of illumination
steps, at the end of which the effective PS II quantum yield as well as
various other parameters are determined with the help of a Saturation
Pulse. Either the ETR parameter or the various Fluorescence
parameters can be displayed. The x-axis corresponds to incident
PAR, as defined by the previously defined PAR list. The current PAR
list can be viewed under Options/PAR-List (see chapter 10). The
definition of ETR is:
ETR = 0.5 x Yield x PAR x 0.84 µequivalents m-2 s-1
(see also section 9.1.1.11). While ETR Light Curves resemble
conventional light response curves, it should be realized that the
illumination periods normally are too short to assure true steady state
conditions. Normally ETR Light Curves are distorted by dark-light
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induction effects. The latter can be minimized by using preilluminated samples.
The maximal ETR reached upon light saturation of
photosynthetic electron flow at high PAR values strongly depends on
correct determination of rather small F values with the help of
saturation pulses. It is important to realize that close to light
saturation an underestimation of Fm' by a few percent will induce a
large underestimation of Y(II) and, hence, also of ETR. In this
context, the Fm factor is important see 9.5.13), which allows to
correct for underestimation of Fm and Fm' due to the unavoidable
heating of the LEDs during a Saturation Pulse. In High Sensitivity or
Microscopy applications using the Special SP-Routine (see 9.6.1)
also the Fm Normalization Factor has a strong influence on correct
assessment of Fm and Fm'.
Fig. 61: Light Curve window showing the Light Curves of two AOIs, for
which the averaged values of the PS II quantum yield parameters
Y(II), Y(NPQ) and Y(NO) are displayed.
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More detailed information on the physiological reactions taking
place during the course of a Light Response Curve is provided by
Light Curves of the various Fluorescence parameters. As illustrated
in Fig. 61, with increasing PAR the Y(II) parameter continuously
decreases, whereas the Y(NPQ) parameter shows an almost
antiparallel increase and the Y(NO) parameter is almost constant.
The curves for the 2 selected AOIs are similar, but not identical. The
three quantum yields always add up to a total of 1. Their relative
values give important information on the partitioning of excitation
energy between photochemical utilization, Y(II), regulated heat
dissipation, Y(NPQ) and unregulated heat dissipation.
For the sake of clarity, in the example of Fig. 62: Report window
showing the Record of the Light Curve recording displayed in Fig.
61 only part of the available fluorescence parameters is displayed.
As outlined above, the user
may choose between display of
ETR
or
of
various
Fluorescence
parameters.
Display
of ten different
fluorescence
parameters is
possible. For display the
corresponding check box has to
be marked. The parameters Fm',
F, Y(II), Y(NPQ), Y(NO),
NPQ/4, qN, qP and qL were
already described in detail (see
section 9.1.1). In principle, all fluorescence parameters
may be displayed on top of each other.
Display of data in the Light Curve window
requires that at least one AOI is selected. If this is not
the case, there is a corresponding warning. A Light
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Curve is started via the Start button and can be terminated at any
time with the help of the Stop button. Recording of a Light Curve
constitutes a New Record. Therefore, unless any preceding Record
already was stored, the user is asked "Save previous Record?".
Furthermore, as Light Curves often are measured with preilluminated samples, the user is asked "Do you want to keep
previously determined Fo, Fm?". During recording of a Light
Curve the number of the current illumination step is indicated in the
Step-box. Next refers to the remaining time until the next
measurement takes place (i.e. application of next Saturation Pulse).
Upon Start automatically the ETR scale limit is
set to 50, which can be either changed manually or
using the Autoscale icon.
Above the Light Curve the time and date of the recording are
documented. The user may also write a short comment into a "text
field", which is saved together with the Light Curve Record. The
text also may be entered or modified in the View-mode. The same
text is automatically also written into a corresponding text field
above the Report file (see 9.4).
After termination of a Light Curve recording the View-mode is
installed. Then the Light Curve Record can be saved using the Save
icon (below Light Curve window).
In the View-mode an extensive analysis of the vast
information stored during a Light Curve can be carried
out.
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There is an infinity of
possible AOIs (at the Image
level) and, hence, in
principle also an infinity of
Light Curves may be
created and analyzed.
When the AOI button
is pressed, the AOI window
is opened, which shows in
its lower part a list of all
AOIs that previously were
defined on the Imagewindow. By clicking a
particular number, the corresponding AOI is selected for data display
in the Light Curve window. The data of several or all AOIs may be
superimposed. In the upper part of the AOI window the data point
symbols of the selected AOI number are shown. Examples are given
for one out of 4 selected AOIs and 8 out of 8 selected AOIs being
active for display, respectively.
When the Editbutton is clicked, a
separate window is
opened, in which the
user may define the
Light
Curve
parameters, i.e. the
number of illumination
steps, the Intensitysetting at each step and
the
time
between
consecutive steps. Up to 20 different illumination steps can be
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defined. In order to modify a current setting (Intensity or Time/10 s),
it first has to be selected by cursor/left mouse click and then the
modified setting has to be entered. Please note that the PAR values
for the selected Intensity settings are derived from the PAR list (see
Options, section 10). It becomes effective with the next mouse click.
When the Uniform time box is checked, the last entered time setting
will be applied for all steps as soon as the Time/10 s cell of another
step is clicked. When the Default button is pressed a Standard Light
Curve featuring 12 Steps is defined, which has proven to give good
results with "normal leaves". This Standard Light Curve is
terminated at intensity setting 16 (approximately 700 µmol quanta
m-2 s-1 PAR). In practice, it rarely makes sense to go beyond this
intensity setting, as the effective quantum yield becomes rather low
and the noise in Yield-determination correspondingly high. In order
to terminate a Light Curve, under Time/10 s a zero (0) has to be
entered.
In some applications it may be of interest to record a Light Curve
with PAR values first increasing until saturation is reached and then
decreasing again, in order to evaluate the capacity of the sample to
recover from light saturation. Such an "Up-Down Light Curve" can
be readily programmed by the user, e.g. by substituting in the Default
list the 0 time value in step 13 by the value 2.
The Light Curve Parameters defined
under Edit can be saved in an lcp-file from
which they can be reloaded at any later time.
In this way, different sets of Light Curve
parameters can be optimized for different
types of plants (e.g. sun and shade plants) and
called up readily without loosing much time.
The lcp-files are saved in the Data-directories of the various
Measuring-Heads.
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Report window
In the Report window the data of the current Record are
displayed in form of listed parameter values. These lists can be
transferred to spread sheet programs, like Excel. At the top of the
Report window there is a text field, into which a comment can be
written. The same text automatically is written into the
corresponding text field on top of the Kinetics or Light Curve
window, if the particular Record was started under Kinetics or Light
Curve. Alternatively, the text can be also written into the
corresponding text field in the Kinetics or Light Curve windows and
then will automatically also appear in the text field above the Report.
Fig. 62: Report window showing the Record of the Light Curve recording
displayed in Fig. 61
Due to the fact that an infinite number of AOIs can be defined by
the user, in principle also an infinite number of Report files can be
derived from a Record stored in a PAM-Image (pim) file. When the
AOI button is clicked the numbers of the presently defined AOIs are
shown. Those AOIs, the data of which shall be displayed on the
Report window, can be selected by left mouse click. They are shown
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in the upper box with their respective symbols in the displays of the
same Record on the Light Curve (or Kinetics) window. Under Select
it is possible to select the fluorescence parameters that shall be listed
in the Report.
Together with a Record also a Comment File can be
saved, which is stored as a txt-file under the same name as
the corresponding pim-file (PAM image).
This comment file can be added or edited at any time in the
View-mode and will be automatically saved as txt-file in conjunction
with the corresponding pim-file. Considering the vast amount of data
which can be collected with the IMAGING-PAM under largely
different measuring conditions, this comment file is of considerable
importance for later assessment of the results.
When the Export icon is clicked, a routine for transfer of
the Report file into an external spread sheet program, like
Excel, is started. Please note that the exported Report corresponds to
the selection of AOIs and fluorescence parameters displayed in the
Report-window.
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After confirmation
by OK the Record
(with the information
specified in the Report
window)
is
first
transferred to the file
export.csv (comma separated values) in the ImagingPam directory.
From there it can be transferred to other programs, like Excel. If
Excel is installed on the PC, the Report-data are automatically
opened under Excel when export.csv in the ImagingPam directory is
double clicked.
At the bottom of the Report window the Settings in abbreviated
form are listed, which apply to the conditions of measurement Nr. 1
(Fo, Fm determination) of the given Record. The meaning of the
abbreviations is as follows:
mi
Measuring light intensity
mf
Measuring light frequency
ai
Actinic light intensity
aw
Actinic width
Icmax
status of Image Correction
g
gain
d
damping
si
saturation pulse intensity
sw
saturation pulse width
bo
booster
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rg
red gain
fmf
Fm-factor
ff
F-factor(not applied under standard settings, see 9.5.14 for
explanations)
fmnf
Fm Normalization Factor
mifo
measuring light intensity for Fo measurement
gfo
gain for Fo measurement
mifm
measuring light intensity for Fm measurement
gfm
gain for Fm measurement
foav
number of Fo averages (the last 6 items apply only for the
MAXI- and MICROSCOPY-versions, if the Special SPRoutine is activated)
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Settings window
The Settings window shows all instrument settings, which can be
modified by the user, and in the Measure-mode also provides
information on the battery status.
Fig. 63: ImagingWin user surface with Settings window being selected
In Fig. 63 the Settings window with standard settings is depicted.
Standard settings can be reinstalled at any time by clicking the
Default Settings button. Most settings relate to light parameters (but
not the light list).
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9.5.1 Light parameters
The IMAGING-PAM features four different types of light:
Measuring Light, Actinic Light, External Light and Saturation
Pulses. Except of the external light all three types of light are derived
from the same source, the LED Illumination Unit (array or single
LED depending on the particular version of the Measuring Head).
The Measuring Light is pulse
modulated. It consists of relatively short (in
the order of 100 µsec) but very short LED
pulses (in the order of 100 µsec). While these
pulses are quite intense, they are applied at a
relatively low repetition rate (frequency) of 1
to 8 Hz and, hence, do not have much actinic
effect. The Measuring Light is automatically
switched on after start of the program. It can be manually switched
off/on via the ML check box (see 8.3). Its Intensity and Frequency
can be set by the user. Standard settings are Intensity 2 and
Frequency 1 (in conjunction with Damping 2) for MAXI-, and
MINI-versions. At these settings the actinic effect of the Measuring
Light is negligibly small. In the case of the MICROSCOPY-versions,
standard settings are Intensity 3 and Frequency 8 (in conjunction
with Damping 5) for the sake of an improved Signal/Noise at low
signal levels. The Meas. Light Int. determines the amplitude of the
fluorescence signal. Normally a signal amplitude of 150 - 200 units
is optimal, assuming maximal stimulation of fluorescence yield
during a Saturation Pulse by a factor of 4 - 5. While signal saturation
occurs at 1000 units, a certain noise band has to be taken into
account which depends on the Damping (see 9.5.2). When dealing
with weakly fluorescent objects (like diluted algae suspensions in
black multiwell plates using the MAXI-version) or in Microscopyapplications a Special SP-Routine is provided, which involves
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automated switching to a high setting of Meas. Light Int. at lowered
Gain-setting. In this way, the Signal/Noise of Fm, Fm', Fv/Fm and
Y(II) measurements can be considerably enhanced. At a given Meas.
Light Int. setting the amplitude of the fluorescence signal can be
adjusted by the Gain (see 9.5.2). Meas. Light Int. up to setting 20 can
be selected. In this way, also weakly fluorescent objects can be
imaged. It should be kept in mind, however, that with objects
showing a light induced fluorescence increase part of this increase
will already occur during an individual Measuring Light pulse, if
Meas. Light Int. is too high. In this case, the F-value will be
overestimated and the saturation pulse induced fluorescence increase
as well as the PS II quantum yield, Fv/Fm or Y(II), will be
underestimated. In principle, it is possible to correct for this effect by
the F Factor (see below).
The Actinic Light drives photosynthesis.
It is switched on manually by the AL check
box or the AL+Y button (see 8.3). The
Intensity and the Width of actinic
illumination can be defined by the user. When
the Width is set to 0, actinic illumination will
not be terminated until manually stopped by
the user by clicking the AL check box. One
out of 20 settings of Act. Light Int. can be selected. The PAR-value
corresponding to a particular setting is shown in the PAR-field when
Actinic Light is switched on (see 8.3). The list of PAR-values
corresponding to all intensity settings can be viewed and edited
under Options/PAR-List (Menu at upper edge of the user surface, see
chapter 10).
Saturation Pulses (SP) are applied for determination of maximal
fluorescence yield (Fm or Fm'). Also the fluorescence yield, Ft,
observed briefly before triggering of the SP is assessed. By
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application of a SP a Measurement is defined, with the resulting
data being saved in the buffer memory. Ten Intensity settings of SP
are available, with the maximal setting 10 being standard. In most
practical applications the best results are obtained with maximal SP
intensity. This, however, is true only, if appropriate use of the Fmfactor correction (see 9.5.13) is made.
During a Saturation Pulse the LEDs are
driven with very high current, which leads to
a temperature increase of the light emitting
chip. As a consequence, the intensity of the
emitted light is transiently lowered by about
5 %. Unavoidably this results in a
corresponding decrease of the intensity of
the Measuring Light, which is driven by the
same LEDs, thus causing underestimation of the Fm value measured
during a Saturation Pulse. This effect can be compensated by the Fm
Factor (see below).
The Width of Saturation Pulses can be changed from 240 to
840 ms (4 x 60 ms to 14 x 60 ms), standard Width of Saturation
Pulses is 720 ms (12 x 60 ms).
When the Booster-Checkbox is activated, the maximal possible
LED-current is applied during the chosen Saturation Pulse width,
which results in a 10 - 15 % increase in Saturation Pulse intensity
beyond the normal intensity at setting 10. The additional LED
heating effect in the Booster-mode is automatically compensated by
a corresponding increase in Measuring Light Intensity for the 800 ms
time period of the Saturation Pulse. While in some application the
Booster may give somewhat higher Fm' values, in the long run the
higher currents will enhance ageing of the LEDs. Therefore
application of the Booster is recommended for special applications
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only. The Booster is not implemented for the MICROSCOPYversions.
9.5.2 Gain and Damping
The Gain determines the amplitude of the
fluorescence signal, Ft, at a given setting of
Measuring Light Intensity (see 9.5.1). Twenty
settings are available, with standard setting 2
for MAXI- and MINI-versions. In the case of
the MICROSCOPY-versions, when dealing
with relatively low signal amplitudes, the Gain
9 is set by default. The Gain should be set such that in the absence of
actinic illumination the fluorescence amplitude (Ft = Fo) is in the
range of 150 - 200 units.
When dealing with weakly fluorescent objects (like diluted algae
suspensions in black multiwell plates using the MAXI-version) or in
MICROSCOPY-applications a Special SP-Routine is provided,
which involves automated switching to a high setting of Meas. Light
Int. at lowered Gain-setting (see 9.6.1). In this way, the Signal/Noise
of Fm, Fm', Fv/Fm and Y(II) measurements can be considerably
enhanced.
The time response of fluorescence measurement can be slowed
down by Damping. The selected Damping-setting determines the
time resolution with which changes of the Ft-image can be viewed
and also the amount of noise visible in the recorded images. Five
settings (0 - 4) are available, with standard settings 2. It has to be
considered that time resolution not only depends on Damping, but on
Measuring Light frequency as well (see 9.5.1). Hence, in order to see
rapid changes in the Ft-image, e.g. while moving a sample, both low
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Damping and high Measuring Light frequency settings have to be
selected. In the Live Video mode (see 9.1.2.3), which applies nearinfrared instead of blue light, Damping is low and Measuring Light
frequency high. Therefore, the Live Video mode is best suited for
positioning samples in the field of view and for focusing images.
9.5.3 Absorptivity
For correct assessment of PAR-Absorptivity the pixel values with
a white piece of paper of the Red image and the NIR image are
supposed to be close to identical (5%) at the given Red and NIR
Intensities (e.g. 0.700). Therefore the NIR and RED image needs to
be adjusted with the NIR Intensity, Red Intensity and Red Gain
settings.
Adjust opens up a prompt for comfortable setting-changes without
switching between the image and the Settings window.
The changes in NIR and Red Settings are recorded in connection
with the Absorptivity-measurement (Measure-Abs. function, see
9.1.2.1 and 9.1.1.10). The Red Gain setting allows fine adjustment
of the intensity of the Red image.
For each instrument suitable NIR and Red Intensities are determined
at the factory and documented together with the corresponding Red
Gain on the LED-Array Illumination Unit. Whereas the steps with
which the LED intensities can be set are relatively coarse, a fine
adjustment via Red Gain is possible. Such adjustment may become
necessary with ageing of the LEDs.
Please note that the NIR Intensity accessible under
Settings/Absorptivity can be set independently from the intensity of
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the NIR light used for measurements of Live Video images (see also
chapter 9.1.2.3).
9.5.4 Slow Induction parameters
The Slow Induction Parameters apply to recordings of
preprogrammed dark-light Induction Curves in
the Kinetics window. After Start and automatic
Fo, Fm determination (see 9.2) some time is
given for fluorescence yield to decline back
close to the original Fo-level, before Actinic
Light is switched on. This Delay-time can be
defined by the user, with the default value being 40 s. The Clocktime determines the time interval between repetitive Saturation
Pulses, with which F and Fm' are measured. At a given length of
actinic illumination, the Clock-time also defines the number of
measurements made in the course of an Induction Curve. The
Duration-time corresponds to the overall length of a recording,
including the Delay-time and the actinic illumination time. The
recording of a standard Induction Curve (under default settings)
involves a total of 15 measurements, with the first corresponding to
Fo, Fm determination and the remaining 14 carried out after start of
actinic illumination.
9.5.5 Image Correction
With the help of Image Correction
unavoidable inhomogeneities of measuring
sensitivity over the imaged area can be
compensated.
One
part
of
such
inhomogeneities originates from spatial
differences in Measuring Light intensity and
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another part is due to the unavoidable vignetting-effect of the
objective lens. For compensation, correction images can be measured
with the help of a sample, which shows uniform fluorescence
emission over the whole imaged area. For this purpose, normal white
printing paper may serve which at high Meas. Light Intensity emits
sufficient fluorescence for a good quality fluorescence image
measured at high Gain setting.
Correction images have to be measured under identical optical
conditions at which the actual experiments are done. In the case of
the MAXI-version this applies particularly to the working distance,
as the inhomogeneities due to Measuring Light intensity are minimal
at 17 - 19 cm distance between the exit plane of the LED-Array and
the sample plane. Using the standard Mounting Stand with Eye
Protection (IMAG-MAX/GS) the working distance is fixed at
optimal 18.5 cm (standard distance). Also the default PAR-list
determined at the factory applies for this standard distance (see 10).
Hence, unless there are compelling reasons to do otherwise, the
standard working distance of 18.5 cm should be used, even when the
Measuring Head is mounted independently from the standard
Mounting Stand (see 3.5).
In the case of the MICROSCOPY-versions the working distance
is determined by the focusing position. As inhomogeneities (due to
dust etc.) will be emphasized in the fully focused position, a position
should be chosen where the image is just out of focus.
With every type of Measuring Head three different correction
images can be stored: Type 1, Type 2 and Maxi (or Mini,
IMAG-L450, RGB).
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For measuring Image Correction please proceed as follows:





set the optical conditions under which the actual measurements
are going to be done (working distance, focusing position, see
above)
select Type 1, Type 2 or Maxi/Mini/IMAG-L470/RGB (under
Settings/Image Correction)
in the case of MAXI- and MINI-versions place at least two layers
of white paper (e.g. folded DIN-A4) into sample plane; in the
case of the MICROSCOPY-version the plastic fluorescence
standard
put the image somewhat out of focus to avoid imaging fine
structures of the white paper tissue or dust etc. on the surface of
the fluorescence standard
press Measure (under Settings/Image Correction)
The measured correction image will be saved until it is
overwritten by a new measurement. The correction images will
remain valid as long as the same optical parameters apply (LED
Illumination Unit, working distance, focusing position, camera
objective lens, microscope objective lens).
While the Image Correction can compensate for heterogeneities
in Measuring Light intensity, the corresponding heterogeneities of
Actinic Light intensity unfortunately cannot be corrected. In
principle, higher PAR values tend to induce lower values of Y(II) and
higher values of Y(NPQ) (see section on Light Curves 9.3). With all
Measuring Heads of the Imaging-PAM M-series maximal deviations
of PAR values from the mean value are small (not exceeding 10 %)
and, hence, can be ignored in most applications.
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9.5.6 Image Transformation
The display of images can be
changed by the Image Transformation
function in order to account for
different positions of the camera with
respect to the user. Images can be
rotated by 180° or mirrored along the vertical midline. Default
settings (Rotate 180° and Mirror boxes not checked) apply for use
of the standard configuration, with the camera pointing downwards
and its wide side pointing towards the user. If, for example, in a
special application the Measuring Head would be pointing upwards,
the shifting of a sample to the left and the right (or up and down)
would cause opposite changes of the displayed image. In this case,
checking Rotate 180° as well as Mirror would be appropriate.
9.5.7 Battery
The Battery-status is indicated by a
blue bar, the height of which represents
the remaining capacity. In addition, the
battery voltage is shown. Please note
that the status display is updated in the Measure-mode only. The
display is updated every minute, except during Light Curve or
Induction Curve recordings. When the Battery Charger 2120-N is
connected, high capacity is indicated even when the battery is only
partially charged. Hence, the true Battery-status should be evaluated
with the Battery Charger being disconnected. When 14.0 V is
reached there is a Low Battery warning. At 13.5 V the instrument is
automatically switched off. The instrument should not be stored with
a discharged battery, which should be recharged every three month.
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Charging of the internal Li-ion battery should be avoided,
when the IMAGING-PAM is switched on, as this may lead
to malfunctioning.
9.5.8 Display parameters
The user may choose between
three different types of image Display.
B/W: black-and-white; gray scale,
ranging from black through shades of
gray to white. This scale normally
provides less contrast than a false color
scale. At the bottom of the scale a cutoff filter is installed, which transforms all pixel values  0.040 into
zero (black) for the sake of background noise suppression (see
below).
Color: standard scale of false colors, ranging from black (pixel
values  0.040) via red, yellow, green and blue to pink (0.999). At
the bottom of the scale a cut-off filter is installed, which transforms
all pixel values  0.040 into zero (black). This filter serves the
purpose to suppress the background noise and to give optimal
contrast between leaf area and background. Even a non-fluorescent
background area gives a weak signal due to unavoidable noise and
some reflected leaf fluorescence. While the position of the cut-off
filter is fixed under Color-display, it can be shifted under Expanded
Color/Analysis (see below).
Expanded Color: Expanded Color can be activated in the view
mode. When selected, the Low and High cut-off limits defined by
the user under Analysis become effective. All pixel values below the
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Low-limit are displayed in black and all pixel values above the Highlimit are displayed in white. For pixel values within the cut-off
limits, the same false color as for the standard Color display is used
(ranging from black via red, yellow, green and blue to purple).
With Low and High limits approaching each other, smaller
differences in pixel values are required to give different colors. This
may help to increase contrast. On the other hand, it is also possible to
lower or completely remove the Low cut-off limit which under
normal Color-display by default is set to 0.040.
False color scale ranging from black via red, yellow, green and blue
to pink with Low and High cut-off limits defined by the user under
Analysis (see 9.1.2.6). All pixel values below the Low-limit are
displayed in black and all pixel values above the High-limit are
displayed in white. With Low and High limits approaching each
other, smaller differences in pixel values are required to give
different colors. This may help to increase contrast. On the other
hand, it is also possible to lower or completely remove the Low cutoff limit which under normal Color-display by default is set to 0.040
(see above).
Fm Scaled Color: The Fm Scaled Color can be activated in the
view mode and serves for emphasizing structures with high
fluorescence yield and suppressing structures with low fluorescence
yield. This can be particularly useful in Microscopy applications
with focused objects displaying high fluorescence yield. As
fluorescence yield depends on the angle of incidence of the
measuring light, Fm Scaled Color images give a 3-D impression.
When Fm Scaled Color is active, the pixel intensities (see info on
Brightness, Settings window upper right corner) are scaled according
to Fm (or Fm’), if the displayed parameter involves assessment of
Fm (or Fm’) with the help of a saturation pulse.
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The fluorescence parameters, images of which can be measured with
the ImagingPam, can be divided into two groups: 1) directly
measured parameters like Fo, F, Fm and Fm’. 2) derived parameters
like Fv/Fm, Y(II), NPQ etc. The latter are based on ratios of the
directly measured parameters and, hence, lack information on the
fluorescence yield. For example, in Microscopy applications a
focused object displaying high fluorescence yield shows the same
Fv/Fm as another object which is out of focus. Display of the latter,
which greatly disturbs the image, is suppressed by the Fm Scaled
Color function, as the Brightness with which it is displayed is low.
This function is automatically installed in the View mode and cannot
be used in the Measure mode.
9.5.9 Go Speed
The Go Speed refers to the rate with
which consecutive images are displayed when
the Go-function is activated in the Viewmode (see 8.2). At maximal speed
spatiotemporal variations of fluorescence parameters can be
presented in a similar way as a video movie. On the other hand,
lower speeds are required to evaluate the observed variations. Please
note that the maximal speed with which calculated parameters like
Y(II) can be displayed, depends on PC processor frequency. For high
speed display of Y(II)-images the Yield Filter (see 9.5.12) should be
inactivated.
9.5.10 PS Limit
The estimated rate of photosynthetic
electron transport, PS, is calculated
according to the equation:
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PS = 0.5 x Y(II) x PAR x Abs. µequivalents m-2 s-1
(see also 9.1.1.11)
In order to display images of this parameter on a false color scale
ranging from 0 to 1, the PS value is divided by a number, which
corresponds to the expected limit of maximal PS, the PS Limit. The
standard setting is 50, which means that the pixel value 1 is reached
when PS/50 = 1. Limits of 50, 100, 150, 200 and 250 can be defined.
9.5.11 Inh. Ref. AOI
The image of the Inh. parameter is
calculated pixel by pixel relative to
the Inh. Ref. AOI, which normally
corresponds to a control AOI. An AOI number between 1 and
100 can be selected. AOI #1 is set by default upon start of the
program.
This parameter is particularly important for assessment of
phytotoxicity with the MAXI-version using multiwell plates.
The Inh. parameter describes the relative inhibition of PS II
quantum yield with respect to a control (see also 9.1.1.18):
Inh. = (Ycontrol - Ysample) / Ycontrol
9.5.12 Yield Filter
The Yield Filter may serve for
suppression of noise in Y(II)-images, which is
mainly due to the noise in the Fm and Fm'
images measured during the relatively short
Saturation Pulses. The Yield-filter is effective in the View-mode
only, i.e. when images are called up from buffer memory (see 8.2).
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Filter settings 0-5 are available, with standard setting 3. Noise
reduction is achieved by averaging the value of every individual
pixel with those of a defined number of neighboring pixels. With
increasing filter setting the number of pixels within an averaged
domain increases (setting 1, 8 neighbors; setting 2, 24 neighbors;
setting 3, 48 neighbors; etc.). This unavoidably leads to some loss in
spatial resolution. Furthermore, depending on the noise structure, the
averaged domains may form patterns which sometimes can be more
disturbing than the original noise. Please note that the Yield-filter
slows down the build-up of the Y(II) image. This may limit the rate
with which consecutive Y(II) images can be displayed using the Gofunction (see 9.5.9).
9.5.13 Fm Factor
The Fm Factor compensates for
underestimation of Fm and Fm' caused by
the decrease of Measuring Light intensity
during a Saturation Pulse, which is related to the unavoidable heating
of the LEDs. In the case of the blue LED-Array Illumination Unit of
the MAXI-IMAGING-PAM, at the standard setting 10 of SP
Intensity this lowering amounts to 5 - 6 %. Hence, in the MAXIversion the standard value of the Fm Factor set by default is 1.055.
For the other versions
different values apply,
that depend on the color
of the applied LEDs.
Red LEDs generally
show
more
heat
induced lowering of
intensity than blue
LEDs. The extent of SP
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induced lowering of ML intensity can be estimated with the help of
the plastic fluorescence standard that is provided with the instrument
(see below). After Reset Settings (see 9.5.15) the Fm Factor is active.
When Fm Factor is active, the Fm (or Fm’) values documented in the
Report (and used for calculation of other fluorescence parameters)
are derived from the product of the actually measured Fm (or Fm’)
and the Fm Factor.
A new Fm Factor can be defined by the user for the current
Record in the Measure as well as in the View mode. In the Measure
mode, however, this definition has to be done before the first
measurement (normally Fo,Fm determination). A corresponding
dialog window is opened by a left mouse click on the current value
in the Fm Factor box. Alternatively this window can be also opened
via Recalc in the Menu (see 10.4). The current value can be erased
and the new value written into the box. The new value is confirmed
and the previously measured data recalculated upon pressing the
Recalc button. The new Fm Factor remains installed until manually
changed or reset to the standard value of 1.055 via the Default
button.
Note:
Recalculation of data on the basis of a new Fm Factor
always applies to the whole Record. If the user tries to
change the Fm factor after Fo, Fm determination, there is a
corresponding warning: "For changing Fm factor start
new record".
The importance of the Fm Factor increases with PAR, as F =
Fm'-F as well as Y(II) = F/Fm' decrease and eventually approach
zero. For example, when at high PAR values the true F approaches
5.5 % of Fm', without this correction the apparent value of F would
approach 0, whereas the true F would approach 0.055. Correct
assessment of such low Y(II) values in any case is problematic due to
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the unavoidable noise limiting Fm' determination. A systematic
underestimation of Fm' and Y(II), however, should be avoided in
view of its pronounced effect on Light Response Curves.
Fig. 64: ETR Light Curve recalculated with Fm Factor 1.000
Fig. 65: ETR Light Curve based on the same original data as in Fig. 64,
recalculated with Fm Factor 1.055
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In Fig. 64 - Fig. 65 the same original Light Curve recording is
displayed after recalculation with Fm Factor = 1.000 (Fig. 64) and
Fm Factor = 1.055 (Fig. 65). With Fm Factor 1.000 maximal ETR
amounts to a relative value of 26 and there is an apparent decline of
ETR at PAR values exceeding 250 µmol quanta m-2 s-1 , which could
be misinterpreted to reflect "photoinhibition". On the other hand,
using the standard value of 1.055, maximal ETR amounts to a
relative value of 31, where the light response curve saturates at about
350 µmol quanta m-2 s-1, without any decline apparent at higher PAR
values. In practice, if there is uncertainty about the correct Fm Factor
to be applied for previously stored data, the data may be recalculated
using several different values. If the chosen Fm Factor is too high, in
a Light Curve this is reflected by a biphasic response without
saturation even at maximal PAR-values.
The actual extent of Measuring Light lowering during a
Saturation Pulse can be estimated with the help of the plastic
fluorescence standard delivered with the instrument. As the
fluorescence yield of this standard in contrast to that of a living leaf
does not change upon illumination with a Saturation Pulse, in first
approximation it may be assumed that the observed decrease of
fluorescence intensity is a measure of the decrease in Measuring
Light intensity. Formally it corresponds to the Fo/Fm or F/Fm' ratio
obtained with the fluorescence standard , which can be calculated
from the values listed in the Report file.
9.5.14 F Factor
The F Factor can be applied for
compensation of the actinic effect of the
Measuring Light pulses, which tends to
cause an overestimation of Fo or F and a corresponding
underestimation of Fv and Y(II). The F Factor is always < 1. It
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corresponds to the factor with which the measured Fo or F value has
to be multiplied in order to obtain correct values. In contrast to the
Fm Factor the F Factor is not an instrument parameter and it cannot
be assumed to be constant. It depends on the physiological condition
of the sample and in particular on the state of PS II reaction centers.
Therefore, the user must decide himself, whether in a particular
application the F Factor correction is advantageous or not. It can not
be recommended to be used in conjunction with Light Curves. The F
Factor check box is not activated upon Reset Settings (see 9.5.15). It
is also possible to recalculate previously recorded data on the basis
of a new F Factor defined by the user.
A new F Factor can
be defined for the
current Record in the
Measure as well as in
the View mode. In the
Measure mode, however, this definition has
to be done before the
first
measurement
(normally Fo,Fm determination). A corresponding dialog window is opened by a left mouse
click on the current value in the F Factor box. Alternatively this
window can be also opened via Recalc in the Menu (see section
10.4). The current value can be erased and the new value written into
the box. The new value is confirmed and the previously measured
data recalculated upon pressing the Recalc button. The new F Factor
remains installed until manually changed or reset to the standard
value of 0.950 via the Default button.
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Recalculation of data on the basis of a new F Factor always
applies to the whole Record. If the user tries to change the
F-factor after Fo, Fm-determination, there is a
corresponding warning: "For changing F factor start new
record".
Two types of actinic effects of the Measuring Light can be
distinguished:
1) Accumulation of closed PS II reaction centers due to repetitive
illumination with Measuring Light pulses. This effect increases
with Measuring Light intensity, pulse frequency and the extent of
dark adaptation of the sample. It does not play any role, when the
overall PAR is high during actinic illumination. Furthermore, in
the case of the Imaging-PAM even at the maximal ML
Frequency there are relatively long dark times between ML
pulses, so that under normal physiological conditions the
accumulation of reduced primary acceptors is insignificantly
small.
2) Closure of a significant fraction of PS II centers during each
individual Measuring Light pulse. This effect increases with
Measuring Light intensity and is favored by a large functional
absorption cross section of PS II. Notably, it also occurs at
minimal pulse frequency of the Measuring Light and does not
become irrelevant during actinic illumination, as long as there
are open PS II reaction centers. In experiments with the ImagingPAM this effect can be quite significant, as relatively strong ML
pulse intensity has to be applied to obtain high quality images at
the given low repetition rates (limited by transmission of large
data volume). The F Factor correction is essential when in
experiments with low fluorescence samples a high Measuring
Light intensity is chosen. In principle, the system sensitivity can
be increased either via higher settings of Gain or Meas.Light Int..
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However at high Gain, also the noise is increased. On the other
hand, at high Meas.Light Int. the overestimation of F (or Fo)
becomes rather large. In first approximation the effect is linear
with intensity. Hence, when an increase from setting 1 to 2
results in an apparent 5 % increase of Fo, this will amount to
about 20 % at setting 5.
The F Factor can be determined by the user for a particular type
of sample and illumination conditions by measuring the F (or Fo) (a)
at Meas. Light Int. setting 1 and high Gain setting (averaging over
several measurements) and (b) at a higher Meas. Light Int. setting
and lower Gain. For comparison, the data first have to be normalized
at Fm' (or Fm). Once the F Factor has been determined, its use
provides an elegant way for high quality imaging of fluorescence
parameters of weakly fluorescing samples, without sacrificing the
correctness of F (or Fo) measurement. However, the proper use of
this correction factor requires some experience and background
knowledge on the physiological background.
9.5.15 Reset Default Settings, Open or Save User Settings
The buttons Default Settings,
Open User Settings and Save
User Settings facilitate the handling of the settings adjustments.
Upon Default settings, the standard settings defined at the factory are
reset. Different standard settings may apply for different types of
Measuring Heads. These settings have proven optimal for imaging of
fluorescence parameters of typical samples using various versions of
the Imaging-PAM. Please note: the Default Settings button does not
reset the PAR-List, Image Correction and Absorptivity adjustments
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The Save and Open User Settings buttons store and open
individually preset settings. Please note these buttons also store or
reopen the active PAR-List!
9.6
High Sens. window
The High Sens. window is implemented for the MAXI- and
MICROSCOPY-versions only. It features a number of functions for
signal enhancement and noise suppression that are quite useful in
applications dealing with weakly fluorescent samples, imaging of
which requires high Gain settings. In the case of measurements with
the MAXI-version using multiwell plates or other objects with
reflecting surfaces the use of the Filter Plate IMAG-MAX/F is
recommended in order to avoid mirror reflections of the LED Array.
Fig. 66: ImagingWin user surface with High Sens. window being selected
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Fig. 66 shows the High Sens. window for the MICROSCOPYversion after enabling the Special SP-Routine (checkbox in upper left
corner). Essentially the same window also applies for the MAXIversion except that the bottom box (Fv/Fm Contrast Enhancement by
Background Suppression) is missing.
9.6.1 Special SP-Routine
Upon start of the program the Special SP-Routine is disabled.
After being enabled via the corresponding checkbox, the Special SPRoutine settings apply. The Special SP-Routine serves for
improvement of signal/noise in measurements of all parameters
involving the application of Saturation Pulses (SP).
Rationale: While Fo or F measurements can be disturbed by high
measuring light (ML) intensities (unintended closure of PS II
centers), Fm or Fm’ measurements always profit from high ML
intensity (closure of PS II centers by SP is intended; higher
signal/noise ratio). With the help of the Special SP-routine ML
intensity can be automatically increased during an SP. To avoid
signal saturation, simultaneously the Gain setting is correspondingly
decreased. The measured Fm or Fm’ values are automatically
corrected by the Fm Normalization Factor, which previously has to
be measured for the selected settings (see under Measure chapter
9.5.13). The Fm Normalization Factor assures that Fm and Fm'
values measured without and with Special SP-Routine are identical.
Fo and F determination: The ML-Int. and Gain settings for
“normal” measurement of fluorescence yield (continuously
monitored Ft) can be selected. Default values are ML3 G9 for
MICROSCOPY- and ML2Gain10 for MAXI-versions. Please note
that the selected settings for ML-Int., Gain, ML-Frequency and
Damping are equivalent to those displayed on the Settings-window.
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Any change of these settings on the High Sens. window will lead to a
corresponding change on the Settings-window and vice versa.
Fm and Fm’ determination: These ML-Int. and Gain settings apply
during the course of the SP only. Default values are ML20G1 for
MICROSCOPY- and ML15G1 for MAXI-versions. At such high
settings of ML-Intensity even weakly fluorescent samples give
satisfactory images and at minimal Gain setting the noise is quite
low.
Measure: For measuring the Fm Normalization Factor, please
replace sample by plastic fluorescence standard delivered with the
instrument, define an AOI and press the Measure button. Then a
measurement of Fo and Fm with the selected Special SP-routine
parameters is carried out. The normalization factor that gives Fm =
Fo with the fluorescence standard is automatically calculated.
Please note that the correct Fm Normalization Factor depends on
the current Fm Factor (see 9.5.13). The same Fm Factor that was
effective during measurement of the Fm Normalization Factor,
should also be effective when this is applied in conjunction with the
Special SP-routine. In contrast the Fm Normalization Factor is not
influenced by the F Factor, as an inert fluorescence standard
different from a living sample does not show any ML pulse induced
fluorescence increase. Therefore, during measurement of the Fm
Normalization Factor the F Factor is automatically disabled.
NOTE:
The user may check on the correctness of the current
Fm Normalization Factor by carrying out a Fo, Fm measurement
with the plastic fluorescence standard using the Special SP-Routine.
As the fluorescence standard does not show any variable
fluorescence, Fo and Fm should be close to identical.
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9.6.2 Fo Averaging
Fo Averaging can be disabled/enabled via the Fo Averaging
checkbox. When enabled, Fo averaging is applied in conjunction
with every Fo, Fm determination. The progress of averaging can be
followed on the Ft image (Image window).
The quality of the Fo images is enhanced by averaging over a
number of images and by selecting a high Damping setting.
Maximal ML Frequency setting 8 is recommended to minimize the
averaging time. Even at maximal frequency the time between ML
pulses is sufficiently long for reoxidation of reduced PS II acceptors.
The time required for a single Fo measurement increases with
Damping setting and amounts to 12 seconds at Damping 5 and ML
Frequency 8.
No of Fo Avg.: Numbers between 1 and 5 can be selected. The time
required for Fo determination is proportional to the number of
averages (e.g. 3 x 12 = 36 seconds with setting 3 at Damping 5 and
ML Frequency 8).
The current setting of No of Fo Avg. also applies for
measurements of RGB images using the MICROSCOPY/RGBversion (see 9.7). This is true irrespectively of whether Fo Averaging
is enabled or not.
9.6.3 Fv/Fm Contrast Enhancement by Background Suppression
This function specifically applies to Microscopy applications
and, hence, is active in the Microscopy-versions of the program only.
It is used in the View-Mode (Measure-checkbox disabled). Please
note that under Settings the Yield Filter checkbox must be enabled.
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Rationale: In epifluorescence microscopy it is unavoidable that
images show some background from scattered / reflected
fluorescence as well as fluorescence of non-focused areas. While
such “unwanted” fluorescence normally is relatively weak and only
marginally disturbs Fo and Fm images, it can strongly affect Fv/Fm
and Y(II) images, which are calculated from ratios of fluorescence
values and, hence, have lost the information on the original signal
amplitudes. This leads to a loss of contrast, so that structures that can
be well discerned in Fo or Fm images may not show in Fv/Fm and
Y(II) images. Fv/Fm pixel associated with low Fm pixel values can
be eliminated by setting an appropriate Fm Limit. In this way the
disturbing effect of a weakly fluorescing background can be
suppressed and the contrast of Fv/Fm images is greatly enhanced.
The same applies to Y(II) images.
Definition Fm Limit: For all pixel displaying Fm values below this
limit, Fv/Fm will be displayed black. The default Fm Limit value is
0.100, which can be reinstalled via Reset.
A new Fm Limit value can be entered after a left mouse click on
the Fm Limit box, which opens a dialog window into which the new
value can be typed and confirmed by O.K.
9.7
RGB-Fit window
The RGB-Fit window is implemented for the MICROSCOPYversion with the Red-Green-Blue LED lamp IMAG-RGB only. It
supports the automated measurement of RGB images and the
consecutive deconvolution into three different pigment types, like
diatoms, green algae and cyanobacteria. For this pupose the RGB
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head uses a red LED with an emission peak at 620 nm, a green
LED (520 nm) and a blue LED with a peak at 460 nm.
Rationale: Deconvolution of fluorescence images into three
different pigment types is based on differences in fluorescence
excitation spectra, similarly as with the PHYTO-PAM Chlorophyll
Fluorometer. For example, cyanobacteria display maximal
fluorescence yield with red-orange excitation (around 620 nm,
phycocyanin absorption) and almost no fluorescence with blue
excitation, as they are lacking Chl b and most of Chl a is associated
with the weakly fluorescing PS I. On the other hand, chlorophytes
and diatoms are characterized by strong fluorescence excitation by
blue light. While diatoms are effectively excited by green (525 nm)
light, green light is distinctly less effective with green algae.
Fig. 67: RGB-Fit window showing RGB-Fit image with deconvoluted
diatoms, chlorophytes and cyanobacteria
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The Measure RGB routine serves for obtaining RGB images that
can be deconvoluted for display of the four different algae groups
(green algae, diatoms, blue-green algae and red algae) in false colors
(green, yellow, blue and red, respectively) with the help of the
preprogrammed fitting routine.
When the Measure button is pressed, automatically Red ML and
Red Image Correction is installed and a Red image is measured. This
measurement involves the averaging of Ft images over 15 s.
Consequently in the same way Green and Blue images are measured.
The pixel intensity values of the obtained images differ from those of
the Ft images, unless the current Red and Blue Gains are 1.000 (see
info text on RGB Gain).
After RGB images are measured, the Fit image is calculated and
displayed. The Fit image is influenced by the current Fit Corr. factors
(see corresponding info text) and the current value of Fit Corr. Avg.
(see corresponding info text). The intensity of a particular pixel is
scaled according to the averaged intensity of the same pixel in the
RGB images.
The time required for measurement of RGB images depends on
the Nr of Fo Avg. set under High Sens./Fo Averaging (see 9.6.2).
Please note that ML Frequency should be at maximal setting 8, as
otherwise RGB measurements take very long. While measurements
of RGB images formally are equivalent to Fo measurements, this
does not mean, that the same low setting of ML Intensity (at
relatively high Gain) is appropriate. Actually, it is recommended to
apply Gain 1, where the maximal signal/noise ratio is obtained and
to increase ML Intensity until sufficiently high fluorescence signals
are observed. For this purpose the user should view the Ft-image on
the Image-window and manually switch between R, G and B
excitation, in order to make sure that fluorescence signals are
appropriate with all three excitation wavelengths.
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9.7.1 RGB Gain
The pixel intensity values of the Red, Green and Blue images are
derived from the pixel intensities of the corresponding Ft images
multiplied by the Red, Green and Blue Gains.
Upon pressing the RGB Gain button a window
upons which shows the current values of Red,
Green and Blue Gains. While the Green Gain is
fixed to 1.000, the Red and Blue Gains can be modified either
manually or via a special Measure routine (see below). Upon
instrument delivery Red and Blue Gains are set to 1.000, as the Red
and Blue ML intensities are adjusted to give appropriate R/G and
B/G ratios for optimal fitting using the standard Axiostar Plus
Epifluorescence microscope with the Fluar 20x/0.75 objective. With
other lenses for optimal fitting the Gains have to be adjusted (see
below).
Defined R/G and B/G ratios are a prerequisite for proper fitting
(deconvolution of differently pigmented algae). Adjustment of RGB
Gain may serve for compensating variations in RGB intensities. Such
variations may occur with ageing of ML LEDs and/or Liquid Light
Guide, choice of different objective lenses etc.
Note
The RGB Gain values have an influence on the RGB images
measured for the purpose of RGB Fit.
9.7.2 Fit Correction
The fit conditions for deconvolution of the 4
major groups of algae are preprogrammed and
are not directly accessible to the user. The Fit
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Correction offers an indirect way to modify the fit conditions.
The deconvolution into the 4 main groups of algae is based on fit
conditions that relate to intensity ratios of individual pixels in the
RGB images. These ratios can be modified by RGB Fit Correction
factors. Fitting then is based on the correspondingly modified RGB
images, with the pixel intensity values of the stored RGB images
being multiplied by the RGB Fit Correction factors.
Upon pressing the Fit Corr. button a window upons which shows the
current values of Red, Green and Blue Fit Corr. factors. While the
Green factor is fixed to 1.000, the Red and Blue factors can be
modified by the user. Upon start of the program all factors are set to
1.000. In this respect the Fit Corr. factors differ from the RGB Gain
factors, which are saved in the Ini-file (see info icon for RGB Gain).
Defined R/G and B/G ratios are a prerequisite for proper fitting
(deconvolution of differently pigmented algae). In practice, even
within the same group of algae there may be some variation of R/G
and B/G signal ratios due to different pigment compositions and
concentrations. Adjustment of Fit Corr. factors may serve for
optimizing deconvolution by modifying the fitting conditions. This
function can be also applied for analysis of saved data in the View
mode. In this respect the Fit Corr. factors differ from the RGB Gain
factors that can be applied in the Measure mode only.
Note
The Fit Corr. factors do not have any influence on the RGB images
measured for the purpose of RGB Fit. This is in contrast to the RGB
Gain factors.
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Definition of AOIs is fully equivalent on RGB-Fit and Imagewindows (see also 9.1.2.2). With the help of the Fit-image it is
possible to define various AOIs that specifically represent particular
organisms (e.g. a diatom attached to a filamentous green alga).
Consequently the photosynthetic performance of these particular
AOIs can be selectively analyzed by measuring various fluorescence
parameters, recording Induction Curves (see 9.2) and measuring
Light Curves (see 9.3).
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10 IMAGINGWIN - Menu Bar
The Menu Bar presents the menu File, Edit, Options, Al-List, Recalc
and Transect including its functions as described in the following
chapter.
10.1 File
10.1.1 Transfer FoFm
In the view mode a FoFm measurement can be transferred to
another .pim file by enabling Transfer FoFm in the file menu. Data
will be recalculated with the given parameter.
10.1.2 Using Skript files - Load Script/Run Script
There are two ways to load an existing or to create a new script
file. The load script command in the file menu or push the load script
file button in the bottom right corner:
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A new window opens up that shows the existing script files:
After choosing a file or entering a new file name and clicking “open”
the editor is started:
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Script File: Open, New, Save, Comment & Back
Four commands are provided for the management of script files
New Script File. The command clears the <Script
File Window> and prompts for a new script file name.
Open Script File. Click to open a stored script file with name
format <filename.PRG>. The default directory for script files
is C:\ImagingPamGigE\Data_MAXI (or respective others
depending on the instruments configuration). Other directories
can be defined using the Windows graphical user interface.
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Save Script File. Saves <Script File> to default or user
defined directory.
Script File Comment. Clicking this icon displays the content
of an editable text file called <filename.TXT> which is
associated with the script file <filename.PRG>.
Editing Tools & Back
Copy Command. The command stores one or several
lines of the current script file into a RAM clipboard. To
execute the copy command, select one or several lines using
the mouse cursor (Left-click once to pick one line). Hold down
<Shift> key and select first and last line of a series of script
file commands to select a group of connected lines. Hold down
<Ctrl> key to select several scattered lines. Click <Copy
Command> icon. The selected commands are now available
for pasting within the current or into another script file using
the <Paste Command>.
Paste Command. To paste previously copied commands,
select a line in the target script file and click the <Paste
Command>. The pasted lines will be written below the
selected line.
Insert Command. To insert a new command in the program
list, mark by a left mouse click a command line in the <Script
File Window>, and then select by left-click in the <Command
Box> the command to be inserted. Clicking the <Insert
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Command> icon will place the new command below the line
marked in the program list.
Delete Command. To delete a command, select one or
several commands as described above and click the <Delete
Command> icon.
Undo Delete Command. Clicking the icon reverses the
last <Delete Command>.
Disable/Enable Command. To disable commands in the
current script file, or enable previously disabled commands,
select commands as described above and click the
<Disable/Enable Command> icon.
In this editor on the left side the possible commands are visible,
on the right hand side the program listing can be seen.
A command can be chosen by clicking on it. The transfer into the
script listing is done by double click on the command or by marking
it and a further click on the red arrow between the both windows .
It is useful to firstly define the basic starting settings of the
experimental script like „Set Meas. Light =“ until „Set Sat. Interval
=“ with which also „personal settings“ can be defined. After the
script has been processed the settings stay at the set values so that
further changes can be done by another script or additional manual
settings.
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For an automated experiment after the basic settings the experimental
settings can be defined:
Programm Commands:
TimeStep(s)
determines a waiting time after the last
command. This command implements the time
needed for the previous command (maybe
800 ms of the FvFm). Using TimeStep the
distortion of a very long script can be avoided.
Wait(s)
determines a waiting time after the last
command independently of the command's
duration.
Call
runs another script file as sub-program, returns
to the running script when the sub-script ends.
Begin of
Repetition Block
initiates a loop – enter the name
the loop and the number of repetitions.
of
Each loop produces one or more lines in the
report so that also the maximum amount of lines
in the report has to be taken into account (max
length of the report is 999 lines) – with e.g. each
saturating flash a new line in the report is
written.
End of Repetition
Block
terminates a loop. Here the number of repetition
is defined.
Paste to Comment enters a remark to the comment line of the report
Line
file.
Message =
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Remark =
to write a remark in the script file. This remark is
not stored in the report file unless the command
"Paste to Comment Line" is chosen.
Spacer
provides an empty row to the script file
Exit
quits IMAGING-WIN
General Commands:
New Record
generates a new record
FvFm
determines the max quantum yield
Yield
starts one saturation pulse (SP) for the
measurement of the effective quantum yield
Ft only
starts a measurement of Ft only
Abs
starts an absorptivity measurement
Measure
switches the measuring light ML on
Start Light Curve
starts a Light Curve
Start Induction
Curve
starts an induction curve
Stop Induction
Curve
stops the induction curve
Save pim file
saves the recorded pim file under a given name
Save NIR file
saves the recorded NIR file under a given name
Export to Tiff File= saves all images as tiff file
Export to CSV File=saves report as csv file
Select Image =
to select exportable image
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Save Tiff Image =
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export selected image as tiff file
Save Jpeg Image = export selected image as jpeg file
General Settings:
Set Gain =
sets gain:
= No to set gain to a desired value
+ or - No to raise or decrease gain by a desired
number of steps
Set Damping =
sets damping:
= No to set damping to a desired value
+ or - No to raise or decrease damping by a
desired number of steps
Light Settings:
Set Meas. Light =
sets measuring light (ML) intensity:
= No to set the measuring light intensity (ML)
intensity to a desired intensity step
+ or - No to raise or decrease measuring light
intensity by a desired number of steps
Set Meas. Freq. =
sets the measuring light frequency
Set Act. Light =
sets actinic illumination (AL) intensity:
= No to set the actinic illumination (AL)
intensity to a desired intensity step (No equals
intensity step of the light list)
+ or - No to raise or decrease actinic
illumination intensity by a desired number of
steps
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Set Act. Width =
Set Sat. Light =
IMAGINGWIN - MENU BAR
sets actinic illumination (AL) width:
= No to set the actinic illumination (AL) width
to a desired duration (in seconds)
+ or - No to raise or decrease actinic
illumination width by a desired number of
seconds
sets saturation pulse intensity:
= No to set the saturation pulse intensity to a
desired value
+ or - No to raise or decrease saturation pulse
intensity by a desired value
Following the whole procedure can be copied by marking the
respective command lines in the listing window and copying with the
button. The button
under the cursor position.
transfers the copied lines to the lines
The diskette symbol button saves the script file with the given name
and the ending “.prg” in the data folder of the PAM instrument.
10.1.3 Exit
Exit quits ImagingWin.
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10.2 Options
10.2.1 Ruler
The Ruler, which shows numbers from 0 to 100, is placed above
the false color scale ranging from 0 to 1. Hence, the ruler may help to
estimate the pixel value of a particular color. For example, yellow
corresponds to pixel values around 0.2.
10.2.2 Scale
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After clicking this option, the Scale window is opened in which
the length of the depicted scale bar can be defined by the user. In the
case of the MAXI-version, for the standard working distance
(18.5 cm) a standard scale width of 19 mm is proposed, which in
principle can be modified by the user. When confirmed via clicking
O.K., the scale width is written underneath the Scale bar. Please note
that the Scale Option can be applied in the View-mode only.
Different standard scale widths are proposed when using the MINIversion (5 mm) and MICROSCOPY-version (0.1 mm). In the case of
the MICROSCOPY-version the scale depends on the particular
microscope and the choice of objective lens. Users are advised to
determine themselves the correct scale for their optical system. The
modified scale width will be saved when the program is closed.
10.2.3 Info Icons
If Info Icons is enabled, information text is available clicking the info
buttons.
10.2.4 Mean over AOI
Two different modes for analysis of the pixel values within an
AOI can be selected under Options.
1) When "Mean over AOI" is enabled, the "Filled" checkbox applies.
2) When "Mean over AOI" is disabled, the "Limits" checkbox
applies.
"Mean over AOI" normally should be enabled with objects for which
AOIs with close to uniform photosynthetic activity can be defined
(e.g. leaf or well filled with algae suspension).
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"Mean over AOI" should be disabled when the photosynthetically
active object is "patchy", so that it is difficult or impossible to define
an AOI with photosynthetic activity (e.g. patches of algae growing in
a well).
Definitions:
Mean over AOI enabled: The pixel values over the whole area of the
AOI are averaged and the average pixel value is displayed.
Mean over AOI disabled: Within the defined AOI only those pixel
values are averaged for which Fv/Fm > 0. Hence, the displayed value
corresponds to areas with photosynthetic activity only.
Filled: When enabled, the whole area of the AOI is "filled" with the
color corresponding to the average pixel value, which works only
when "Color" is selected under Settings/Display. When disabled,
each pixel is displayed with the color corresponding to its individual
pixel value.
Limits: When enabled, the pixel values in the photosynthetically
active areas are averaged, whereas the non-active areas are displayed
in black. In this way the limits of the photosynthetically active areas
with respect to the non-active areas are emphasized.
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Fig. 68: Mean over AOI enabled (A) and disabled (B)
10.2.5 Define AOI-array geometry
After clicking "Define AOI-array
geometry" a window is opened in
which the number of rows and lines
of the array grid can be defined.
After confirmation by O.K. this
definition will remain valid until a
new definition is carried out. Before
an AOI array can be created, the
position of this array within the
overall image has to be defined. This
is done by definition of the AOIs in
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the upper left and lower right corners of the array.
10.2.6 Create AOI array:
After the two corner AOIs are defined, the Create AOI-array
Option can be carried out and the corresponding array will be
displayed after some delay (calculation time).
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In the above example an array of 24 AOIs with 4 rows and 6
lines in the center of a 96 well microtiter plate was defined. While all
wells were filled with aliquots of algae suspension, only the 24 wells
covered by the array contained increasing concentrations of the PSII
inhibitor diuron, which is reflected by decreasing values of Y(II).
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10.3 Al-List
In the AL-List menu actinic light lists can be loaded, viewed and
edited via LED currents / PAR values. In combination with an
Ulm-500 Light Meter & Logger also a Light Calibration routine is
available.
10.3.1 LED currents / PAR values
LED currents / PAR values
opens up the window displayed on
the left.
"Open PAR-file" loads an existing
.par file.
"Save as PAR-file" saves the
displayed PAR values in a .par file
and gives the opportunity to save a
comment file in the context of this
PAR-list.
"Show comment file" displays the
information stored in conjunction
with the opened .par file.
The Help button provides further
information.
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The PAR-List shows the LED current and PAR values for the
Actinic Light intensity settings 0 - 20. LED Current as well as PAR
values may be edited. Possible Current values range from 1 to 511.
Changes in LED current values result in changes in LED light
intensity. PAR values are listed in µmol quanta m-2 s-1. Editing the
PAR values does not change the LED intensities, it changes the
annotation of the actinic Light intensity. Different lists apply to the
various Measuring Heads. In the case of the MAXI-version, the
Default PAR-List refers to the standard working distance of 18.5 cm
between LED-Array Illumination Unit and sample plane. The listed
values can be edited by the user and the edited list confirmed by
clicking O.K.. A default.par PAR-List for each measuring head
version, determined at the factory under standard conditions, is
stored in the corresponding Data folder.
While the relative values of Actinic Light intensities 0 - 20 do
not vary between individual instruments, there may be some
variation between instruments in terms of absolute intensities due to
different charges of LEDs. Absolute Intensities can be determined in
conjunction with an ULM-500 Light Meter & Logger by the light
calibration routine described in the next chapter.
In microscopy applications the PAR does not only depend on the
LED intensity, but also on the choice of objective lens. Using the
RGB-Head also the LED color has to be taken into account.
Calibration can be done using an ULM-500 with the microquantum
sensor (e.g. MC-MQS) or the use of relative instead of absolute PAR
values may be recommended. In any case, when dealing with
microscopically small organisms, there is a large difference between
incident and absorbed PAR.
When dealing with leaves, it has to be considered that as a
consequence of pronounced light gradients within the samples, the
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effective PAR at a particular setting does not correspond to one
value, but rather to band of values with Gaussian distribution around
a center value. Therefore differences between incident and effective
PAR values may be expected. On the other hand, as most of the
measured fluorescence originates from the leaf surface, the observed
light response will be dominated by the upper cell layers where the
incident intensity is effective. These aspects have to be considered
when comparing apparent electron transport rates derived from
fluorescence measurements (ETR) with gas exchange rates. In
contrast to chlorophyll fluorescence, the gas exchange response
involves deeper cell layers with effective PAR being attenuated
relative to incident PAR.
10.3.2 Light Calibration
Light Calibration requires an ULM-500 Light Meter & Logger with
a microquantum sensor e.g. LS-C for the MAXI- and MINI- version
or MC-MQS for the MICROSCOPY-version.
Please connect ULM-500 to the computer before starting
ImagingWin. "Ulm active" appears in the window title to show
enabled ULM-500 - ImagingWin communication.
Clicking Light Calibration in
the Al-List menu opens up a
Light
Calibration
window.
Please place the microquantum
sensor at the object´s position
and
start
the
automated
calibration routine for the 20 light intensities by pressing Start.
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10.4 Recalc
With the help of the Recalc Option it is
possible to recalculate the data of a given Record on
the basis of correction factors for Fm (or Fm') and F
as already described in the sections on Fm Factor (9.5.13) and F
Factor (9.5.14). Recalculation is always for the whole Record. It can
be carried out in the View- as well as in the Measure-mode. In the
Measure mode, however, this definition has to be done before the
first measurement (normally Fo,Fm determination). If the user tries
to change the F factor after Fo, Fm-determination, there is a
corresponding warning: "For changing Fm factor start new
record" or "For changing F factor start new record".
Recalculation is started upon clicking the Recalc button. When
the Default button is clicked, the data are recalculated on the basis
of the Default value of the Fm Factor = 1.055 or the Default value of
the F Factor = 0.950. As the Recalculation is carried out in the Viewmode, it is irrelevant whether under Settings the Fm Factor and F
Factor checkboxes are enabled or disabled.
The possibility of recalculation of previously recorded data in the
View-mode is particularly helpful, if at the time of the actual
measurements there is uncertainty about the proper values of Fm and
F Factors. In the case of Light Curves, for example, the data may be
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recalculated on the basis of various Fm Factors to obtain a
reasonably shaped saturation curve. A biphasic response curve
without apparent saturation suggests that the applied Fm Factor is too
high. If the response curve shows a maximum followed by a decline,
this normally suggests that the applied Fm Factor is too low. Unless
rather long illumination times at high intensity settings are applied, a
genuine decline of ETR by photoinhibition is unlikely.
Application of a F Factor in conjunction with a Light Curve is
problematic and cannot be recommended. As F values are increasing
with PAR, the relative increase of F due to the individual ML pulses
(see 9.5.14) will decline. Therefore, application of a constant F
Factor is bound to result in overestimation of ETR at high PAR. If
Light Curves were recorded with a F Factor being enabled, the data
can be readily recalculated with F Factor = 1.
10.5 Transect
The Transect Option allows to plot the
pixel values of an imaged parameters along a
previously defined line segment. In this way a
profile of this parameter across a sample can be obtained.
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Upon clicking Define Endpoints a dialog window is opened in
which the user may choose one out of 5 line widths and define the
coordinates of the two endpoints of the line segment. With
increasing line width the signal/noise ratio of the Transect plot is
increased. The coordinates of endpoint 1 (X1, Y1) and endpoint 2
(X2,Y2) can be manually entered into the corresponding boxes. Then
upon O.K. the Transect profile is displayed. If the coordinates of the
envisaged endpoints are not known, as normally the case, the
endpoints can also be defined graphically. When the Graphic button
is clicked, in the Image window a line segment appears, one of the
endpoints of which temporarily is fixed, while the other one can be
freely moved with the cursor (marked by a cross) until it is fixed by a
left mouse click. Now the other end is marked by the cursor and can
be freely moved to the second endpoint position, which can be fixed
by another left mouse click. Then automatically the Transect profile
is calculated and displayed in a separate window.
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The Pixel values of the selected Image parameter are plotted as a
function of Pixel number along the line segment. In the lower left
corner of the window the x-y coordinates of the two endpoints of
the transecting line segment are displayed. They may serve for
reproducing an identical transect at a later time. In the lower right
corner the Pixel value and Pixel number of the point marked by the
cursor are displayed. When the Export button is clicked, a list of all
Pixel value/Pixel number couples is saved in form of the file
profile.csv into the Data directory of the applied Measuring-Head,
from where it can be exported into a spread sheet program, like
Excel. If Excel is installed on the PC, the export.csv file is
automatically opened under Excel. Please note that the export.csv
file will be overwritten with the following data export. To avoid this
it should be renamed.
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LIST OF KEY COMMANDS
11 List of key commands
The IMAGING-PAM normally is operated via the ImagingWin
user surface by cursor and mouse click operations. However, also
some key commands are possible which in certain instances may be
used as shortcuts. All key commands require simultaneous pressing
of the Ctrl-key:
Ctrl A
Actinic Light on/off
Ctrl I
Open Image window
Ctrl K
Open Kinetics window
Ctrl L
Open Light Curve window
Ctrl M
Fo, Fm determination
Ctrl Q
Switch between Measuring Light Frequency 1 and 8
Ctrl R
Open Report window
Ctrl S
Open Settings window
Ctrl V
Switch to Live Video mode
Ctrl Y
Yield determination by Saturation Pulse
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12 Technical specifications
12.1 Components used in all Versions
12.1.1 Control Unit IMAG-CG
Control Unit IMAG-CG
Design:
Aluminum housing featuring large size
built-in Li-ion battery, sockets for cable
connections with CCD Camera IMAG-K6
or IMAG-K7, connectors for the MAXI-,
MINI- MICROSCOPY-Measuring Heads,
an external light source (EXT) and Battery
Charger 2120-N
Microcontroller:
RISC processor
User interface:
Pentium PC with ImagingWinGigE
Software; connection via GigE ethernet;
keyboard operation; monitor screen display
Power supply:
Internal rechargeable Li-ion battery
14.4 V/6 Ah
Power consumption:
9 W (500 mA) drawn from internal Li-ion
battery
Recharging time:
approx. 4 hours (IMAGING-PAM turned
off) via Battery Charger 2120-N
Operating temperature:-5 to +45 °C
Dimensions:
25 cm x 10.5 cm x 11 cm (L x W x H)
Weight:
2.1 kg (incl. battery)
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12.1.2 IMAG-K7
Design:
CCD Chip size:
Interface:
Dimensions:
Weight:
Black and white C-mount camera operated
in 10-bit-mode at 30 frames/sec featuring
1/2" (640 x 480 pixels)
GigE-Vision®
8,64 cm x 4,4 cm x 2,9 cm (L x W x H)
(without objective lens)
<200g
12.1.3 IMAG-K6
Design:
CCD Chip size:
Interface:
Dimensions:
Weight:
Black and white C-mount camera operated
in 10-bit-mode at 30 frames/sec featuring
2 x 2 pixel binning
2/3" (1392 x 1040 pixel primary
resolution)
GigE-Vision®
8,64 cm x 4,4 cm x 2,9 cm (L x W x H)
(without objective lens)
<200g
12.1.4 Windows Software ImagingWin
Minimum PC requirements: processor 1.7 GHz, 4 GB free RAM,
built-in Gigabit Ethernet (GigE), Windows
Vista, Windows 7 or 8
Features:
Data display and instrument settings on up
to 7 different windows
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 Image: display
parameters
of
18
different
 Kinetics: time dependent changes of
fluorescence parameters
 Light
Curve:
registration
of
preprogrammed light response curves
 Report: numerical lists of parameter
values for selected areas of interest
 Settings: instrument settings
 High Sens.: routines for enhanced
signal/noise and contrast (MAXI- and
MICROSCOPY-versions)
 RGB-Fit: deconvolution of 3 different
pigment
types
(e.g.
diatoms,
chlorophytes
and
cyanobacteria)
(MICROSCOPY/RGB-version)
12.1.5 Battery Charger 2120-N
Input:
90 to 264 V AC, 47 to 63 Hz
Output:
19 V DC, 3.7 A
Operating temperature: 0 to 40 °C
Dimensions:
15 cm x 6 cm x 3 cm (L x W x H)
Weight:
300 g
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12.2 Components specifically relating to Maxi-version
12.2.1 LED-Array Illumination Unit IMAG-MAX/L
LED-Array mounted on printed-circuit
board in aluminium housing with central
opening for CCD Cameras IMAG-K6 or
IMAG-K7 with miniature ventilator and
cable connections to Control Unit IMAGCG and External 300 W Power Supply
(chapter 12.2.4)
Light source for fluorescence excitation and actinic illumination:
44 royal-blue 3 W Luxeon LEDs (450 nm)
equipped with individual collimating
optics; standard excitation intensity
0.5 µmol quanta m-2 s-1 PAR, modulation
frequency 1-8 Hz; max. actinic intensity
1200 µmol quanta m-2 s-1 PAR; max.
saturation pulse intensity 2700 µmol
quanta m-2 s-1 PAR
Light sources for assessment of absorbed PAR:
16 red LEDs (660 nm); 16 NIR LEDs
(780 nm)
Working distance:
standard 18.5 cm for 9 cm x 12 cm image
area; at 22.5 cm distance 11 cm x 15 cm
image area (mounted independently of
IMAG-MAX/GS on separate stand)
Light field properties: Vertical incidence on sample; LED
distribution optimized for uniformity; at
standard working distance maximal
deviation from mean intensity +/- 7 %
Dimensions:
18.5 cm x 18.5 cm x 4.5 cm (L x W x H)
Design:
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Weight:
TECHNICAL SPECIFICATIONS
1.3 kg (incl. cable 1 m long)
12.2.2 LED-Array Illumination Unit IMAG-MAX/LR
LED-Array mounted on printed-circuit
board in aluminium housing with central
opening for CCD Cameras IMAG-K6 and
IMAG-K7, filter plate IMAG-MAX/FR
and miniature ventilator and cable
connections to Control Unit IMAG-CG and
External 300 W Power Supply (chapter
12.2.4)
Light source for fluorescence excitation and actinic illumination:
44 red 3 W Luxeon LEDs (650 nm)
equipped with individual collimating
optics; standard excitation intensity
0.5 µmol quanta m-2 s-1 PAR, modulation
frequency 1-8 Hz; max. actinic intensity
1200 µmol quanta m-2 s-1 PAR; max.
saturation pulse intensity 2700 µmol
quanta m-2 s-1 PAR
Light sources for assessment of absorbed PAR:
16 red LEDs (660 nm); 16 NIR LEDs
(780 nm)
Working distance:
standard 18.5 cm for 9 cm x 12 cm image
area; at 22.5 cm distance 11 cm x 15 cm
image area (mounted independently of
IMAG-MAX/GS on separate stand)
Light field properties: Vertical incidence on sample; LED
distribution optimized for uniformity; at
standard working distance maximal
deviation from mean intensity +/- 7 %
Dimensions:
18.5 cm x 18.5 cm x 4.5 cm (L x W x H)
Design:
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Weight:
TECHNICAL SPECIFICATIONS
1.3 kg (incl. cable 1 m long)
12.2.3 Optional filter plate IMAG-MAX/F (only for IMAG-MAX/L!)
Design:
Filter properties:
Dimensions:
Weight:
Black-anodized aluminium plate with 44
individual blue filters to be mounted in
front
of
collimating
optics
of
IMAG-MAX/L
1 mm blue-green glass filters (BG 39,
Schott) blocking red transmission and
passing 90 % of blue light
186 mm x 176 mm x 2.5 mm (L x W x H)
180 g
12.2.4 External 300 W Power Supply
Input:
90 to 264 V AC, 50/60 Hz
Output:
43 to 57 V, 5.2 A
Operating temperature: 0 to 40 °C
Dimensions:
226 mm x 110 mm x 58 mm (L x W x H)
Weight:
1.75 kg
12.2.5 K7-MAX/Z
Design:
Cosmicar-Pentax zoom objective lens
(F1.0/f=8-48 mm) and close-up lens,
extension ring, detector filter (RG645,
3 mm) and short pass interference filter (λ
< 770 nm); adapter-frame for mounting
CCD-camera MAG K7 at increased
working distance.
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Dimensions:
Weight:
Filter Screw Size:
Mount:
Focal Length:
Max. Aperture Ratio:
Iris Range:
Temperature Range:
57 x 95 mm
430 g (without adapter-frame)
φ55 mm, P=0.75 mm
C-Mount
8 to 48 mm
1: 1.0 (f=8 to 28) to 1.2 (f=48)
F/1.0 to F/22
-20°C to +50°C
12.2.6 K7-MAX/S
Design:
Dimensions:
Weight:
Filter Screw Size:
Mount:
Focal Length:
Iris Range:
Temperature Range:
Cosmicar-Pentax
objective
lens
(F1.2/f=12 mm), extension ring and
detector filter (RG645, 3 mm) and shortpass interference filter (λ < 770 nm).
30 x 35.5 mm
67 g
M27 x 0.5
C-Mount
12 mm
F/1.2 to F/22
-20°C to +50°C
12.2.7 K6-MAX
Design:
Dimensions:
Weight:
Filter Screw Size:
Mount:
192
Cosmicar-Pentax
objective
lens
(F1.4/f=12.5 mm), detector filter (RG665,
3 mm) and short-pass interference filter (λ
< 770 nm).
42 x 50 mm
135 g
M40.5 x 0.5
C-Mount
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TECHNICAL SPECIFICATIONS
Focal Length:
Iris Range:
Temperature Range:
12.5 mm
F/1.4 to C
-20°C to +50°C
12.2.8 K6-MAX/M and K7-MAX/M
Design:
mounting set IMAG-K6 or IMAG-K7
camera on IMAG-MAX/L or IMAGMAX/LR. Consisting of camera holder and
metal rod 15 cm length, 15 mm diameter.
12.2.9 Mounting Stand with Eye Protection IMAG-MAX/GS
Design:
Sample position:
Dimensions:
Weight:
Aluminium frame featuring two clamps for
mounting LED-Array Illumination Unit
IMAG-MAX/L or IMAG-MAX/LR; red
perspex sliding hood for eye protection;
removable bottom part
Detached leaves, slides or petri dishes
resting on x-y stage for variable
positioning; special frame for defined
positioning of multiwell plates; after
removal of bottom part, possibility to jack
up whole Mounting Stand for study of
plants in pots or trays
23.5 cm x 24.5 cm x 22.5 cm (L x W x H)
3.1 kg
193
CHAPTER 12
TECHNICAL SPECIFICATIONS
12.2.10 IMAG-MAX/B
Design:
Aluminium frame featuring two clamps for
mounting LED-Array Illumination Unit
IMAG-MAX/L or IMAG-MAX/LR
Sample position:
Detached leaves, slides or petri dishes
resting on x-y stage for variable
positioning; fix working distance 18.5 cm
in a plane perpendicular to the optical axis.
Dimensions:
18.8 cm x 17.8 cm x 20.4 cm (L x W x H)
Weight:
455 g
12.2.11 ST-101
Design:
Laboratory stand with wooden baseplate
for mounting LED-Array Illumination Unit
IMAG-MAX/L or IMAG-MAX/LR
Dimensions:
40 cm x 30 cm x 70 cm (L x W x H),
Weight:
455 g
12.2.12 Transport Box IMAG-MAX/T
Design:
Dimensions:
Weight:
194
Aluminum box with custom foam packing
for
MAXI-IMAGING-PAM
and
accessories
60 cm x 40 cm x 25 cm (L x W x H)
5 kg
CHAPTER 12
TECHNICAL SPECIFICATIONS
12.2.13 IMAG-MAX/GWK1
Design:
Adapter Plate with legs and eye protection
for positioning IMAG-MAXI Head on
3010-GWK1
Dimensions:
18.5 cm x 20 cm 17 cm (L x W x H)
Weight:
856 g
12.3 Components specifically relating to MINI-version
12.3.1 IMAG-MIN/B
Design:
12 Luxeon LEDs 460 nm with individual
short pass filters and collimator optics; 16
red 650 nm and 16 NIR 780 nm LEDs for
measuring PAR-absorptivity; max. actinic
intensity, 2000 µE/m²s; max. Saturation
Pulse intensity, 6000 µE/m²s; frame at
fixed working distance (7 cm); for imaging
24 mm x 32 mm sample area; suitable for
use in combination with IMAG-K6 and
IMAG-K7 with camera accessories
(objectives).
Dimensions:
Weight:
11.8 cm x 9,4 cm x 8.6 cm (L x W x H)
552 g (incl. cable)
195
CHAPTER 12
TECHNICAL SPECIFICATIONS
12.3.2 IMAG-MIN/R
Design:
12 Luxeon LEDs 620 nm with individual
short pass filters and collimator optics; 16
red 650 nm and 16 NIR 780 nm LEDs for
measuring PAR-absorptivity; max. actinic
intensity, 2500 µE/m²s; max. Saturation
Pulse intensity, 7500 µE/m²s; frame at
fixed working distance (7 cm); for imaging
24 mm x 32 mm sample area; suitable for
use in combination with IMAG-K6 and
IMAG-K7 with camera accessories
(objectives).
Dimensions:
Weight:
11.8 cm x 9,4 cm x 8.6 cm (L x W x H)
552 g (incl. cable)
12.3.3 IMAG-MIN/GFP
Design:
12 Luxeon LEDs 480 nm with individual
short pass filters and collimator optics; 16
red 650 nm and 16 NIR 780 nm LEDs for
measuring PAR-absorptivity; max. actinic
intensity; frame at fixed working distance
(7 cm); for imaging 24 mm x 32 mm
sample area; only suitable for use in
combination with IMAG-K6 with camera
accessories K6-MIN, K6-MIN/FS and K6MIN/M.
Dimensions:
Weight:
11.8 cm x 9,4 cm x 8.6 cm (L x W x H)
552 g (incl. cable)
196
CHAPTER 12
TECHNICAL SPECIFICATIONS
12.3.4 K7-MIN
Design:
Dimensions:
Filter Screw Size:
Mount:
Focal Length:
Iris Range:
Temperature Range:
Weight:
Cosmicar-Pentax
objective
lens
(F1.4/f=16 mm) detector filter (RG645,
3 mm) and short-pass interference filter ( λ
< 770 nm), extension ring 4.2 mm
30 x 33 mm
M27 x 0.5
C-Mount
16 mm
F/1.4 to 22
-20°C to +50°C
58 g
12.3.5 K6-MIN
Design:
Dimensions:
Filter Screw Size:
Mount:
Focal Length:
Iris Range:
Temperature Range:
Weight:
Cosmicar-Pentax
objective
lens
(F1.4/f=25 mm) detector filter (RG645,
3 mm) and short-pass interference filter ( λ
< 770 nm), extension ring 7.2 mm
30 x 37.3 mm
M27 x 0.5
C-Mount
25 mm
F/1.4 to 22
-20°C to +50°C
76 g
12.3.6 K6-MIN/FS
Design:
for parallel imaging of GFP- and Chlfluorescence of identical sample areas
197
CHAPTER 12
TECHNICAL SPECIFICATIONS
(instead of camera objective accessory
detector filter RG645, 3 mm and short-pass
interference filter λ < 770 nm). Featuring
alternative detector filters for green or red
fluorescence (special green filter set 500 575 nm and red filter RG665, 3 mm with
short-pass interference filter λ < 770 nm).
12.3.7 K7-MIN/M and K6-MIN/M
Design:
devices for mounting camera IMAG-K6 or
IMAG-K7 to the Imaging MINI-Heads
Weight:
166 g
12.3.8 IMAG-S
Design:
Fine drive laboratory stand with high
performance rack-and-pinion drive (50 mm
traverse path) for adjustment of working
distance; platform base with covered 9.3
cm central hole for mounting optional
sample holders
Weight:
3.34 kg
12.3.9 IMAG-MIN/ST
Design:
198
Fine drive tripod adapter for mounting a
MINI-Head onto a tripod head with UNC
1/4-20 screw threads, High perfomance
rack-and-pinion drive (120 mm traverse
path) for adjustment of working distance
CHAPTER 12
TECHNICAL SPECIFICATIONS
12.3.10 ST-1010
Design:
Compact Tripod, for tripod heads with
UNC 1/4-20 screw threads.
12.3.11 IMAG-MIN/BK
Design:
Leaf clip mountable on sample frame of all
MINI-Heads; MINI-Head holding grip
Weight:
184 g
12.3.12 IMAG-MIN/GFS
Design:
Adapter plate with snap-on-mount for
connecting IMAG-MINI Head to Standard
Measuring Head 3010-S,
Dimensions:
9.5 cm x 6 cm x 1.4 cm (L x W x H)
Weight:
30 g
12.4 Components specifically relating to MICROSCOPYversions
12.4.1 IMAG-AXIOSCOPE
Design:
Modified
AxioScope.A1
Microscope
(Zeiss) adapted for IMAGING PAM
applications. Comprises binocular fototube
(30°/23 100:0/0:100), condenser 0,9/1,25H
and transmitted light unit HAL 50.
Detector filter RG665, dichroic mirror 420199
CHAPTER 12
TECHNICAL SPECIFICATIONS
640 nm, video adapter 60N-C 2/3" 0,5x
and standard lens Fluar 20x are already
mounted.
12.4.2 IMAG-L470M
Design:
Microscope LED Lamp Module 470 nm
(blue) for fluorescence excitation of Chl
fluorescence of most algae groups
including cyanobacteria. Emission peak at
470 nm. Shipped together with a set of
neutral grey filters for using the system
together with higher magnifications.
12.4.3 IMAG-L625M
Design:
Microscope LED Lamp Module 625 nm
(red-orange) for fluorescence excitation of
Chl fluorescence of most algae groups
including cyanobacteria. Emission peak at
625 nm. Shipped together with a set of
neutral grey filters for using the system
together with higher magnifications.
12.4.4 IMAG-RGB
Design:
200
Red-Green-Blue Microscopy LED Lamp
allowing computer-assisted deconvolution
of major algae groups. Fluorescence
excitation and actinic illumination using
red (620 nm), green (520 nm), blue (460
CHAPTER 12
TECHNICAL SPECIFICATIONS
nm) or white light (mixed 620, 520 and
460 nm); featuring fluid light guide
(100 cm length, 3 mm active dia.),
connecting to collimator optics at
excitation
port
of
epifluorescence
microscope; with cable to be connected to
RGB output socket at IMAG CG; featuring
printed circuit board with separate drivers
for RGB LEDs;
12.4.5 IMAG-AX-REF
Design:
Reflector Module with filter set for
additional microscope LED lamp modules.
Consisting of a beam splitter filter (420640 nm) and a detector filter (665 nm),
mounted in a Zeiss reflector module frame
Technical specifications are subject to change without prior notice.
201
CHAPTER 13
WARRANTY CONDITIONS
13 Warranty
All products supplied by the Heinz Walz GmbH, Germany, are
warranted by Heinz Walz GmbH, Germany to be free from defects in
material and workmanship for two (2) years from the shipping date
(date on invoice).
13.1 Conditions
This warranty applies if the defects are called to the attention of
Heinz Walz GmbH, Germany, in writing within two (2) years of the
shipping date of the product.
This warranty shall not apply to
- any defects or damage directly or indirectly caused by or
resulting from the use of unauthorized replacement parts and/or
service performed by unauthorized personnel.
- any product supplied by the Heinz Walz GmbH, Germany which
has been subjected to misuse, abuse, abnormal use, negligence,
alteration or accident.
- to damage caused from improper packaging during shipment or
any natural acts of God.
- to batteries, cables, calibrations, fiberoptics, fuses, gas filters,
lamps, thermocouples, and underwater cables.
Submersible parts of the DIVING-PAM or the underwater version of
the MONITORING-PAM have been tested to be watertight down to
the maximum operating depth indicated in the respective manual.
Warranty shall not apply for diving depths exceeding the maximum
operating depth. Further, warranty shall not apply for damage
202
CHAPTER 13
WARRANTY CONDITIONS
resulting from improper operation of devices, in particular, the
failure to properly seal ports or sockets.
13.2 Instructions to obtain Warranty Service,
Please follow the instructions below:
The Warranty Registration form must be completed and returned to
Heinz Walz GmbH, Germany.
The product must be returned to Heinz Walz GmbH, Germany,
within 30 days after Heinz Walz GmbH, Germany has received
written notice of the defect. Postage, insurance, and/or shipping costs
incurred in returning equipment for warranty service are at customer
expense. Duty and taxes are covered by Walz. Accompany shipment
by the Walz Service and Repair form available at:
http://www.walz.com/support/repair_service.html
All products being returned for warranty service must be carefully
packed and sent freight prepaid.
Heinz Walz GmbH, Germany is not responsible or liable, for missing
components or damage to the unit caused by handling during
shipping. All claims or damage should be directed to the shipping
carrier.
203
CHAPTER 14
INDEX
14 Index
2 2120-N
B 10, 27, 46
A Abs.
97, 107, 137
absolute intensities
179
Absorptivity 76, 97, 107, 137
Actinic illumination
71
Actinic Light
87
AL
87
AL + Y
88
Allied Vision Techn.
17
Allied Vision Technologies 15
Analysis
113
AOI
68, 108, 118
AOI-array
110, 175
aperture
16, 46
Apoplan
51
apparent rate of
photosynthesis
99
Area of Interest
68
Autoscale
121
Axio ScopeA.1
50, 58
204
B/G
160
Background Suppression 154,
156
Battery Charger
10, 27, 46
beam splitter
52, 53
BG39
28
binning
16
Buffer Memory
116
C camera
Capture
CCD camera
charge
Charge LED
chlorophytes
Clock
Clock-time
components
Components
connecting the cables
Contrast Enhancement
Control Unit IMAG-CG
coordinates
Correction file
10
113
9
10
10
158
89
138
27
46
63
154
9
183
138
CHAPTER 14
Create AOI-array
current fluorescence
Cursor
INDEX
176
91
113
D dark adaptation
92
dark fluorescence parameters
69
dark fluorescence yield
91
dark-adaptation
69, 93
dark-recovery
120
deconvolution
157
Default
181
Default Settings
152
Define AOI-array geometry
175
Delay-time
138
diatoms
157
distance rods
42
Duration-time
138
E effective PS II quantum yield
94
electron transport rate
100
Endpoints
183
energy dissipation
71
Epifluorescence Microscope
46
ETR
76, 100
event marker
121
Expanded Color
113
Export
86, 129
Export button
184
Ext
87
Ext. out
10
External Light Source
87
eye protection
14, 19, 25
F F 92
F Factor
149, 155, 181
false color code bar
68
Filter Plate
12
filter slider
34
Filters
57
first measurements
66
fit correction
160
Fluar
50
Fluorescence Mode
110
fluorescence parameters 118
fluorescence yield
68, 92
Fm
70, 83, 92
Fm'
93
Fm Factor
146, 155, 181
Fm Limit
157
Fo
70, 83, 91
Fo Averaging
156
Fo Avg.
159
Fo, Fm-determination 83, 94
Fo'-determination
104
205
CHAPTER 14
INDEX
Ft-image
67
Fv/Fm
83, 93
Fv/Fm Contrast Enhancement
154, 156
Fv/Fm-image
70
G gas exchange
Genty
getting started
GFP detection
GFP MINI Head
GFS-3000
GFSWin
GigE cable
GigE-Vision®
Go
Go Speed
Graphic button
grip holder
42
94
63
35
34
42
43
32
15, 17
86
74, 144
183
29, 38
H High Sens.
High Sens. window
159
153
I Illumination Unit
IMAG-CG
206
11, 12
9, 27, 46
image capture and analysis 90,
107
Image Correction 20, 44, 138,
140
image intensity
18
Image Transformation
141
imaged area
20, 24
Image-window
74, 90
ImagingWin
27, 43, 46, 64
ImagingWin software
81
IMAG-K6 15, 30, 31, 46, 47
IMAG-K7
17, 18, 30, 31
IMAG-L365M
46
IMAG-L470
47
IMAG-L470M
10, 46
IMAG-L625
47
IMAG-L625M
10, 46
IMAG-MAX/F
12, 22
IMAG-MAX/FR
12
IMAG-MAX/GS 13, 14, 19
IMAG-MAX/GWK
26
IMAG-MAX/L
11, 12
IMAG-MAX/L
10
IMAG-MAX/L
16
IMAG-MAX/LR
12, 16
IMAG-MAX/T
8, 21
IMAG-MIN/B
27, 28
IMAG-MIN/BK
27, 38
IMAG-MIN/GFP
27, 32
IMAG-MIN/GFS
42
IMAG-MIN/R
27, 28
IMAG-PC
25
CHAPTER 14
IMAG-RGB
Ind. Curve
Ind.+Rec.
Induction Curves
Inh.
Inhibition
Installation
software
Introduction
INDEX
46, 47
119
120
74
106
106
64
3
J JEPG
JPEG
86
74
31
17
18
31
73
116
73
L Lake model
lcp-file
Leaf Clip
Leaf Holder
LED drivers
56
11, 12
23
16
110
180
87
183
67
M K K6-MIN/M
K7-MAX/M
K7-MAX/Z
K7-MIN/M
Kautsky effect
Kinetics window
Kinetics-window
LED Modules
LED-Array
legs
lens aperture
Life Video
Light Calibration
light controls
line widths
Live Video
95, 96
127
29, 37
27, 39
9
Manual recording
119
MAXI-HEAD
12
Maximal fluorescence yield 92
Maximal PS II quantum yield
93
MAXI-version
7
Mean over AOI
173
Measure
84
Measure Abs.
76, 107
measure RGB
159
Measuring Light
87
memory
25
microcontroller
9
Microscopy LED Lamp
46
MICROSCOPY-version 46,
154
MINI-Head
10, 28, 31
MINI-version
27
Mirror
141
mirroring
22
207
CHAPTER 14
mirroring samples
ML
Mounting Stand
Multi Control Unit
multiwell plate
INDEX
12
87
19
27
12, 22
N near-infrared
67
New Record
70, 84
NIR Intensity
137
NIR LEDs
97
NIR light remission
100
NIR-light
67
NIR-measuring
67
NIR-remission
76
noise mask
94, 96, 97
noise suppression
153
non-photochemical
103
Notebook
25
NPQ
83, 101
O objective
Off-line mode
Open
operating system
Options
Overload box
208
16
74
86
25
90
111
P PAM Image (PIM)-file
83
PAR-Absorptivity 11, 29, 30,
33, 76, 98, 107
PAR-List
44, 88, 179
PC
25
photochemical quenching 104,
105
photosynthetic parameters 68
Photosynthetically Active
Radiation
88
phytotoxicity
106
pim
74
pixel
15, 184
Pixel number
184
pixel value
184
Plan-Apochromat
50
Power on/off
9
power supply
9
processor
25
profile.csv
184
PS
78, 99
PS II quantum yield
70
PS/50
79, 99
Q qL
105
qN
83, 103
qP
83, 104
quantum sensor 88, 179, 180
CHAPTER 14
INDEX
quantum yield of
nonregulated energy
dissipation
96
quantum yield of regulated
energy dissipation
95
quenching coefficients
69
R R/G
160
Rapid Light Curves
74
Recalc
181
Record
83, 128
Red Gain
137
red light remission
102
reflection
22
Reflector Modules
51
register cards
81
report window
128
reset
109, 113, 157
RG 645
18, 30
RGB-Fit window
157
RGB-Head
10, 61
RISC processor
9
Rotate 180°
141
R-remission
76
Ruler
90
Saturation Pulse
69, 87, 88
Save
86
Scale
172
Screw Jack
23
Setup files
65
Show commend
86
signal enhancement
153
signal/noise ratio
159
Skript commands
165
Skript programming
163
software
27
software installation
64
Special SP-Routine
92, 93,
154
start icon
66
Stern-Volmer
95, 96, 102
Switching the detector filter 33
synchronous operation
43
T technical specifications 186
TIFF
74, 86
Transect
182
Transport Box
21
types of images
91
U S Safety instructions
SAT-Pulse
1
88
Ulbricht Sphere
updates
USB interface
76
65
25
209
CHAPTER 14
User Settings
INDEX
152
x-y coordinates
x-y stage
184
21
V video adapter
View-mode
vignetting
46
84, 85
14
W working distance 14, 20, 24,
29, 42, 139, 173
Y Y(II)
Y(NO)
Y(NPQ)
Yield Filter
94
94, 96
94, 95
145, 156
Z Zoom
18, 112
X X-Y
121
 Heinz Walz GmbH, 2014
Heinz Walz GmbH  Eichenring 6  91090 Effeltrich  Germany
Phone +49-(0)9133/7765-0  Telefax +49-(0)9133/5395
E-mail [email protected]  Internet www.walz.com
210
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