PHOTOSYNTHESIS YIELD
ANALYZER MINI-PAM
Portable Chlorophyll Fluorometer
Handbook of Operation
2.115 / 04.96
2. Edition: August 1999
minip_1eb.doc
© Heinz Walz GmbH, 1999
Heinz Walz GmbH • Eichenring 6 • 91090 Effeltrich • Germany
Phone +49-(0)9133/7765-0 • Telefax +49-(0)9133/5395
E-mail info@walz.com • Internet www.walz.com
Printed in Germany
CONTENTS
1 Safety instructions ........................................................................ 1
1.1 General safety instructions ........................................................ 1
1.2 Special safety instructions......................................................... 1
2 General Information .................................................................... 2
3 Basic Operation of the MINI-PAM............................................. 4
4 Description of the eight Keyboard Functions ............................ 7
4.1 Single key operations ................................................................ 7
4.2 Double key operations............................................................... 8
5 Important Points for Correct YIELD-Measurements ............ 10
6 Description of the Memory-Function ....................................... 12
7 The Mode-Menu ......................................................................... 14
7.1 List of Menu points ................................................................. 15
7.2 Description of the Mode-menu points..................................... 17
8 Components of the MINI-PAM................................................. 29
8.1 Main Control Unit ................................................................... 29
8.1.1
Fluorescence excitation and detection .............................. 30
8.1.2
Special information on MINI-PAM/B .............................. 31
8.1.3
Internal halogen lamp as actinic light source .................... 33
8.1.4
Rechargeable battery......................................................... 34
8.1.5
LC-display......................................................................... 35
8.1.6
Electronic components ...................................................... 36
8.1.7
Description of the connectors ........................................... 37
8.2 Fiberoptics MINI-PAM/F and Miniature Fiberoptics
MINI-PAM/F1 ........................................................................ 38
8.3 Leaf-Clip Holder 2030-B ........................................................ 41
8.4 Micro Quantum/Temp.-Sensor 2060-M.................................. 45
8.5 External Halogen Lamp 2050-HB........................................... 45
8.6 Dark Leaf Clip DLC-8 ............................................................ 47
I
CONTENTS
9 Data Transfer.............................................................................. 48
10 Operation of the MINI-PAM via a PC-Terminal and the
RS 232 Interface ......................................................................... 51
11 Maintenance................................................................................ 53
11.1 Internal battery and its replacement ........................................ 53
11.2 Halogen lamp and its replacement .......................................... 54
11.3 Fuse replacement..................................................................... 55
11.4 EPROM and its replacement ................................................... 55
12 Chlorophyll Fluorescence Measurements with the
MINI-PAM.................................................................................. 57
12.1 Chlorophyl fluorescence as an indicator of photosynthesis .... 58
12.2 The PAM measuring principle ................................................ 64
12.3 Assessment of photosynthesis with the MINI-PAM: Outline
of the most important functions in practical applications. ...... 67
12.3.1 Maximal photochemical yield Fv/Fm ............................... 67
12.3.2 ML-BURST (menu point 5).............................................. 68
12.3.3 AUTO-ZERO (menu point 2) ........................................... 69
12.3.4 Fo, Fm (menu point 25) .................................................... 70
12.3.5 INT.TEMP (menu point 35).............................................. 71
12.3.6 qP, qN and NPQ (menu points 26 and 27)........................ 73
12.3.7 YIELD-measurements of illuminated samples ................. 75
12.3.8 ACT-LIGHT and ACT+YIELD (menu points 12 and 13) 76
12.3.9 LIGHT CURVE (menu point 17) and LIGHTCURVE+REC (menu point 18) ........................................ 79
12.3.10 YIELD- and ETR-averaging (menu point 11) .................. 82
12.3.11 INDUCTION CURVE (menu point 21) and
INDUCTION CURVE+RECOVERY (menu point 22).... 83
12.3.12 Repetition Clock (menu point 28: REP-CLOCK and
double key function ON+MEM)....................................... 85
II
CONTENTS
13 Appendix ..................................................................................... 87
13.1 Technical specifications .......................................................... 87
13.2 List of warnings and errors...................................................... 91
13.3 PIN-assignments...................................................................... 93
13.4 List of commands for operation of MINI-PAM via
PC-terminal by user-written software ..................................... 94
13.5 Selected reviews on chlorophyll fluorescence and related
topics ....................................................................................... 98
14 Rechargeable battery ............................................................... 104
15 Warranty conditions ................................................................ 105
III
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.
Ensure that no liquids or other foreign bodies can find their way
inside the device.
10. The device should only be repaired by qualified personnel.
1.2
Special safety instructions
1. The MINI-PAM Photosynthesis Yield Analyzer 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.
2. The MINI-PAM employs high intensity light sources which may
cause damage to the eye. Avoid looking directly into these light
sources during continuous illumination or saturation pulses.
1
CHAPTER 2
GENERAL INFORMATION
2 General Information
The Photosynthesis Yield Analyzer MINI-PAM has been
developed with special attention to the quick and reliable assessment
of the effective quantum yield of photochemical energy conversion
in photosynthesis. The most relevant information is obtained by a
single key operation within a second and up to 4000 data sets can be
stored for later analysis. Due to a novel opto-electronic design and
modern microprocessor technology, the MINI-PAM is extremely
compact and at the same time highly sensitive and selective. It is
ideally suited for rapid screening of photosynthetic activity in the
field, green house and laboratory and due to its robust, waterproof
housing it can be used even in extreme environments.
The MINI-PAM, like all PAM Fluorometers, applies pulsemodulated measuring light for selective detection of chlorophyll
fluorescence yield. The actual measurement of the photosynthetic
yield is carried out by application of just one saturating light pulse
which briefly suppresses photochemical yield to zero and induces
maximal fluorescence yield. The given photochemical yield then
immediately is calculated, displayed and stored. Numerous studies
with the previously introduced PAM Fluorometers have proven a
close correlation between the thus determined YIELD-parameter
(ΔF/Fm') and the effective quantum yield of photosynthesis in leaves,
algae and isolated chloroplasts. With the help of the optional LeafClip Holder 2030-B also the photosynthetic active radiation (PAR)
can be determined at the site of fluorescence measurement, such that
an apparent electron transport rate (ETR) is calculated. In addition to
this central information, the MINI-PAM also provides the possibility
of measuring fluorescence quenching coefficients (qP, qN, NPQ),
applying continuous actinic light for measurement of induction
curves (Kautsky-effect) and automatic recordings of light-saturation
2
CHAPTER 2
GENERAL INFORMATION
curves with quenching analysis. For these purposes, an extensive
MODE-menu is provided.
While the MINI-PAM was conceived as a typical stand-alone
instrument for field experiments, it can also be operated under
laboratory conditions in conjunction with a PC and the special
Windows-software WinControl. When the MINI-PAM is connected
to a PC, the information on instrument settings and data registration
is continuously exchanged, such that both ways of operation are
equivalent.
The WinControl software provides so-called "tooltips" with short
explanations of the numerous functions of the MINI-PAM. Hence,
use of WinControl is recommended particularly to the beginner for
becoming acquainted with the principles of operation and of
chlorophyll fluorescence information. It should be emphasized that
there is no risk of serious mistakes causing damage. Hence,
beginners may feel free to "play" with the system, trying out all
functions. For this purpose, the Chart-window is particularly useful,
as it records all fluorescence changes like a chart recorder.
This manual deals mainly with the MINI-PAM as such, operated
as a stand alone unit. A separate manual will be provided for the
WinControl software.
3
CHAPTER 3
BASIC OPERATION OF THE MINI-PAM
3 Basic Operation of the MINI-PAM
The MINI-PAM is very easy to operate. It has a two-line LCdisplay and a small tactile keyboard with eight function keys (ON,
OFF, MODE, MEM, ∧, ∨, START, SET). In order to get started, only
the fiberoptics have to be connected and the ON-key is pressed. Now
the system is ready for recording fluorescence yield of any sample
which is close (5-20 mm) to the free end of the fiberoptics. The
actual measurement of the most relevant YIELD-parameter (quantum
yield of photochemical energy conversion) just involves pressing the
START-key. Then on the display, for example, the following
information is shown:
1:
F: 448
445F 1739M
745Y
..E
..C
..L
The meaning of the various displayed parameters is as follows:
1:
Number denoting the standard MODE-menu position 1
which is automatically installed whenever the MINI-PAM
is switched on or a YIELD-determination is carried out
via START.
445F
Fluorescence yield (F) measured briefly before the last
saturating light pulse triggered by START.
1739M
Maximal fluorescence yield (M = Fm or Fm') measured
during the last saturating light pulse triggered by START.
..C
Temperature in degree Celsius, display of which requires
optional Leaf-Clip Holder 2030-B.
F: 448
Momentary
fluctuations.
4
fluorescence
yield
displaying
small
CHAPTER 3
BASIC OPERATION OF THE MINI-PAM
745Y
The most relevant YIELD-parameter determined by the
last saturating light pulse triggered by START, calculated
as follows:
YIELD = Y/1000 = (M-F)/M = ΔF/M = ΔF/Fm'
(Genty-parameter)
With a dark-adapted sample ΔF/Fm = Fv/Fm,
corresponding to the maximal yield of photochemical
energy conversion.
..E
Relative rate of electron transport (ETR), display of
which requires optional Leaf-Clip Holder 2030-B. It is
calculated by the formula:
ETR = E = YIELD x PAR x 0.5 x ETR-factor
..L
Light intensity in terms of PAR (quantum flux density of
photosynthetically active radiation), display of which
requires Leaf-Clip Holder 2030-B.
After every operation of START the obtained data set with the
corresponding time and date is entered into a RAM-memory, with a
storage capacity of 4000 data sets. The stored data can be called on
the display via the MEM-key. Previously recorded data can be
recalled by using the arrow-keys (∧ or ∨). Stored data can be printed
out via an RS 232 interface or transferred on a PC for further
analysis.
The MINI-PAM has been pre-programmed at the factory with
standard settings (see list in 7.1) for all relevant measuring
parameters (for example Measuring Light Intensity, Gain, Damping,
Saturation Pulse Intensity, Saturation Pulse Width etc.). These
standard settings are optimized for measurements with standard leaf
samples at approx. 12 mm distance between fiberoptics and leaf
surface. For special applications, there is great flexibility for
appropriate adjustment of all measuring parameters with the help of
the extensive MODE-menu, using the arrow-keys (∧ and ∨) in
5
CHAPTER 3
BASIC OPERATION OF THE MINI-PAM
combination with the SET-key. Details are given in the MODE-menu
list (see 7.2).
6
CHAPTER 4
DESCRIPTION OF KEYBOARD FUNCTIONS
4 Description of the eight Keyboard Functions
Fig. 1: Photosynthesis Yield Analyzer MINI-PAM
4.1
ON
Single key operations
To switch MINI-PAM on (short pressing of the key).
To activate the backlighting of the display (switches
automatically off when no key operation for 50 s; power
saving for field use); requires 3 s pressing of the key.
OFF
To switch MINI-PAM off; will occur automatically, if no
key operation for 4 min (power saving for field use), unless
disabled via menu point 10.
MODE To return to MODE-menu after using the MEM- or SETkeys.
7
CHAPTER 4
DESCRIPTION OF KEYBOARD FUNCTIONS
MEM
To enter the MEMORY-level of stored data with the last
stored data set being displayed.
∧, ∨
To select one of 51 points of the MODE-menu or one of
4000 data sets when MEMORY is activated:
To change a particular parameter setting in the MODEmenu after operating the SET-key.
For advancement by several steps these keys can be kept
pressed.
START To trigger a saturating light pulse for assessment of YIELD
and related fluorescence parameters.
To start and stop selected function.
SET
4.2
Double key operations
Besides the single key operations, there is a number of double
key operations which can serve as short-cuts for selecting/carrying
out certain items/commands in the MODE-menu. For this purpose,
the first key must be kept firmly pressed before briefly pressing the
second key.
MODE+START To return to standard display (menu position 1).
MODE+SET
To move from one functional block in the MODEmenu to the next (see list in 7.1).
MODE+∧
To move to MODE-menu point 17: LIGHT CURVE
(carried out via SET).
MODE+∨
To move to MODE-menu point 21: IND.CURVE.
MODE+ON
To switch measuring light on/off.
MODE+MEM
To move to MODE-menu point 28: REP-CLOCK.
8
CHAPTER 4
DESCRIPTION OF KEYBOARD FUNCTIONS
ON+SET
To switch actinic light on/off.
ON+START
To start/stop actinic illumination
measurement (see menu point 13).
ON+MEM
To start/stop the clock for repetitive triggering of
selected function (e.g. saturation pulses when 29:
CLOCK-ITEM in position SAT).
ON+∧
To start/stop a LIGHT CURVE (equivalent to menu
point 17).
ON+∨
To start/stop an INDUCTION CURVE (equivalent to
menu point 21).
SET+OFF
To reset program, if MINI-PAM for some reason
does not respond to key-operations.
with
yield-
If the MINI-PAM is switched on by RS 232-access the keycontroller may not respond. In this case push the ON-key once.
Note:Whenever a command is given which involves the switching
on and off of the actinic halogen light source, a short beepsound confirms that the command is carried out. In addition,
there is a more extended beep for the duration of a saturating
light pulse.
9
CHAPTER 5
IMPORTANT POINTS
5 Important Points for Correct YIELDMeasurements
The main purpose of the MINI-PAM is the reliable determination
of the YIELD-parameter ΔF/Fm (Genty-parameter). This task is
carried out by the MINI-PAM with exceptional sensitivity and
reproducibility. Because of the central importance of this particular
type of measurement, a special section is devoted to it in this
handbook (see section 12.3). Here just the most important practical
aspects are outlined which are essential for correct YIELDmeasurements:
1) The distance between sample and fiberoptics should be approx.
10-15 mm, such that a normal leaf at standard settings gives a
signal of 200-500 units.
2) The AUTO-ZERO function (MODE-menu point 2) should be
applied (while sample is removed), in order to suppress any
unavoidable background signal which otherwise would cause
some lowering of the YIELD-reading (see 12.3.3).
3) In practice, YIELD-measurements make sense only, if the light
conditions of the sample are well controlled. For example, a leaf
may be severely damaged in Calvin cycle activity and still show a
high YIELD-value when dark-adapted or in weak light. The
overall photosynthetic performance should be assessed during
steady state illumination at a photon flux density which is
somewhat below saturation in a control sample. For highest
accurracy it is essential that the PAR is measured close to the spot
of the sample where also fluorescence is detected. For this
purpose the optional Leaf-Clip Holder 2030-B is available (see
8.3). On the basis of the measured YIELD- and PAR-data an
apparent electron transport rate (ETR) is calculated and displayed
10
CHAPTER 5
IMPORTANT POINTS
(...E). The plot of ETR vs. PAR corresponds to a light-response
curve of photosynthesis (see 12.3.9).
4) When YIELD is measured under field conditions, it is essential
that the leaf position and effective PAR are not inadvertently
changed. During the actual measurement, the fiberoptics must be
stably fixed with respect to the leaf surface for ca. 2 s, e.g. with
the help of the Leaf-Clip Holder 2030-B.
5) Dark YIELD-measurements require special conditions (see also
12.3.1). As already pointed out in 3), such measurements cannot
give information on the overall photosynthetic performance. They
are useful to specifically assess the state of PS II, for example
following light stress treatment. In this case, it is essential, that
the measuring light does not induce any significant increase of
fluorescence yield. For this purpose, the MODE-menu point 5
provides the possibility of applying the measuring light in short
pulse bursts, thus cutting its integrated intensity to 1/5 (see
12.3.2).
11
CHAPTER 6 DESCRIPTION OF THE MEMORY-FUNCTION
6 Description of the Memory-Function
All data recorded via START are automatically stored in RAMmemory with a capacity of 4000 data sets. They can be recalled on
display via the MEM-key. Then, for example, the following
information is shown:
MEM 382: 12:27 27/MAY/95
A:
322Y 21.1E 157L
In the top line it can be seen that the data set Nr. 382 of the
current MEMORY was recorded at 12:27 o'clock on May 27th 1995.
The bottom line shows that a sample of type A was used (see
MODE-menu point 51), which displayed a YIELD-value (Y) of
0.322 and an apparent ETR-value (E) of 21.1 at an incident light
intensity (L) of 157 μmol quanta m-2 s-1 of the photosynthetically
active radiation (PAR).
More information relating to this particular data set can be
displayed in the top line by SET-operation:
MEM 382:390F 576M 19.9C
A:
322Y 21.1E 157L
After the first SET, the top line shows that the fluorescence yield
(F) measured briefly before the saturating light pulse was 390, that
the maximal fluorescence (M) amounted to 576 and that temperature
was 19.9 °C.
MEM 382:645P 759N 1.557Q
A:
322Y 21.1E 157L
After the second SET, the top line shows the quenching
coefficients qP=0.654, qN=0.759 and NPQ=1.557, which will be
meaningful only if for this particular sample a Fo-Fm determination
12
CHAPTER 6 DESCRIPTION OF THE MEMORY-FUNCTION
(MODE-menu point 25) had been carried out beforehand (see
12.3.4).
Further operation of SET (2x) leads back to the original display
with time and date.
Using the arrow keys ∧ and ∨ one can move within the memory
and display any previously recorded data sets.
All data stored in MEMORY can be cleared by the CLEAR
MEMORY function (MODE-menu point 39). For safety's sake, this
command does not only require execution by SET, but in addition
confirmation by the ∧-key. The memory is organized in form of a
ring storage and its clearance normally is not required, as old data
will be automatically overwritten.
The MEMORY-front normally corresponds to the MEM-No.
under which the last set of data was stored. It can be moved to any
number between 1 and 4000 with the help of MODE-menu point 38.
After any change in instrumental settings, the complete set of
settings will be stored upon the next YIELD-measurement in the
Report-file of the WinControl program (see separate manual). This is
indicated by "Saved Settings" in the MEMORY-display.
Data stored in MEMORY can be readily transferred to a PC via
the RS 232 cable (see section 9).
13
CHAPTER 7
THE MODE-MENU
7 The Mode-Menu
The MODE-menu contains 51 items corresponding to a variety
of measured values, instrumental settings or special commands. The
positions of the various menu points were arranged for optimal
practicability, with the most frequently used functions being closest
to the standard position 1.
Increasing or decreasing position numbers are selected by the ∧or ∨-arrow keys, respectively. Changes are terminated via SET or
MODE. Starting from position 1, at increasing numbers there are
mostly MODE-points involving commands (for example, 2: AUTOZERO), while at decreasing numbers the MODE-points for
instrumental settings prevail (for example, 50: MEASURING
LIGHT INTENSITY). Some of the MODE-menu positions can be
directly reached via double key operations (see list in section 4.2
above).
Irrespective of the selected menu position, a YIELDmeasurement can be initiated at any time by pressing the START-key.
Normally, the system then automatically returns to the menu position
1 where the measured data set is displayed. The only exceptions are
menu-positions 11, 25-27 and 34, where the displayed values are of
primary interest.
The operations related to the various points of the MODE-menu
are either directly carried out via SET (e.g. 2: AUTO-ZERO: 50) or
initiated/terminated (e.g. 50: MEAS-INT: 8) by pressing SET.
Settings are changed by arrow key operations (∧, ∨) and become
immediately effective. The numbers following the double points
show the present settings.
14
CHAPTER 7
7.1
THE MODE-MENU
List of Menu points
The Menu points are organized in functional blocks. The starting
point of each block can be reached successively by simultaneous
pressing of MODE and SET. The frequently used positions MARK,
MEAS-INT and GAIN can be readily selected by going backwards
from position 1 using the ∨-key.
The below list shows the default settings, which can be reset at
any time by the command 36: RES. SETTINGS. The first points of
the functional blocks which can be quickly reached by the
MODE+SET command, are emphasized by boldface printing. The
double-key commands by which some of the menu points can be
quickly accessed are also listed.
Menu points:
1. Standard display
2. AUTO-ZERO:
0(SET)
3. MEAS.LIGHT: ON (SET)
4. M.FREQ: LOW (SET)
5. ML-BURST: OFF(SET)
6. LIGHT AV15s:OFF(SET)
7. EXT.LIGHT-S:ON (SET)
8. LIGHT CALIB:
(SET)
9. DISP.ILLUM.:OFF(SET)
10. AUTO-OFF: ON (SET)
11. AV. YIELD and ETR
12. ACT-LIGHT: OFF(SET)
13. ACT+YIELD: OFF(SET)
14. ACT-WIDTH 0:30 (SET)
15. ACT-INT: 5 (SET)
16. AL-FACT: 1:00 (SET)
17. LIGHT CURVE:OFF(SET)
Quick access via:
MODE+START
MODE+ON
MODE+∧
15
CHAPTER 7
THE MODE-MENU
18. L.CURVE+REC:OFF(SET)
19. LC-WIDTH 0:10 (SET)
20. LC-INT: 3 (SET)
21. IND.CURVE: OFF(SET)
22. IND.C+REC: OFF(SET)
23. IND-DELAY 0:40 (SET)
24. IND-WIDTH 0:20 (SET)
25. Fo and Fm (SET)
26. qP and qN (SET)
27. NPQ (SET)
28. REP-CLOCK: OFF(SET)
29. CLOCK-ITEM:SAT(SET)
30. CLK-TIME:00:30(SET)
31. TIME 17:32:56 (SET)
32. DATE 17-OCT (SET)
33. YEAR 1997
(SET)
34. BATT: 12.4V (11.8)
35. INT.TEMP: 23C
36. RES.SETTINGS: (SET)
37. PROGR.D2.07(280698)
38. MEMORY: 12 (SET)
39. CLEAR MEMORY (SET)
40. LIGHT-OFFS: 0(SET)
41. LIGHT-GAIN:1.00(SET)
42. TEMP.OFFS: 0.0(SET)
43. TEMP.GAIN: 1.00(SET)
44. ZERO-OFFS: 20(SET)
45. ETR-FAC: 0.84 (SET)
46. SAT-WIDTH:0.8s(SET)
47. SAT-INT: 8 (SET)
48. DAMP: 2
(SET)
16
MODE+∨
MODE+MEM
CHAPTER 7
49. GAIN: 2
50. MEAS-INT:
51. MARK: A
7.2
THE MODE-MENU
(SET)
8 (SET)
(SET)
Description of the Mode-menu points
The following list briefly describes the items contained in the
MODE-menu, some of which are outlined in more detail in section
12.3 (Assessment of photosynthesis yield with the MINI-PAM).
Standard settings are shown.
Standard menu-position for display of the
data measured by last saturating light pulse
triggered by START. The 4 central parameters F, M, Y and E, the
present fluorescence signal F: (with blinking *), temperature (°C)
1:
F: 448
445F 1739M 19.9C
745Y 6.2E
20L
and and ambient PAR (L) are displayed.
Command for determination of signal in
absence of sample (background signal), the
value of which is displayed and automatically subtracted, such that
signal becomes zero without sample. This offset value remains
effective for all following measurements until being deliberately
changed. It has to be newly determined whenever 50: MEASURING
LIGHT INTENSITY or 49: GAIN are modified. If this is not done
there is a warning ?NEW OFFSET? when YIELD is determined by
START. The warning will stop when a new offset is determined via
menu point 2 or the given offset is confirmed in menu position 1 via
SET.
2: AUTO-ZERO: 20(SET)
F: 448 745Y 6.2E
20L
On/off switch of measuring light. Under
standard conditions the measuring light is
on. When switched off, a negative signal indicates the AUTO-ZERO
value (see menu point 2). The switch can also be operated via
MODE + ON without entering the MODE-menu.
3:MEAS.LIGHT: ON (SET)
F: 448 745Y 6.2E
20L
17
CHAPTER 7
THE MODE-MENU
Switch between the standard measuring
pulse frequency of 0.6 kHz (LOW) and 20
kHz (HIGH). At 20 kHz the signal/noise is increased by a factor
of 5-6. On the other hand, at this high frequency the measuring light
intensity can induce substantial fluorescence changes. Hence, 20 kHz
normally should be used only when its actinic effect can be
neglected relative to a stronger ambient light (e.g. above 100 μmol
quanta m-2 s-1).
4: M.FREQ:
F: 448 745Y
LOW (SET)
6.2E
20L
Switch between normal signal detection
(continuously pulsed measuring light) and
signal detection by short bursts of measuring light. In the latter case,
pulse trains are 0.2 s with dark-intervals of 0.8 s, resulting in a
reduction of integrated measuring light intensity by a factor of 5.
This can be advantageous for assessment of the maximal
photochemical yield after dark-adaptation (ΔF/Fm = Fv/Fm). In the
ML-BURST mode the basic frequencies of 0.6 or 20 kHz are
maintained.
5: ML-BURST: OFF(SET)
F: 448 745Y 6.2E
20L
When this function is enabled, the readings
of the external light sensor are averaged
over a period of 15 s, in order to account for fluctuations of light
intensity. It is important that the sensor remains fixed in a given
position for 15 s before the actual measurement of quantum yield.
6:LIGHT AV15s:OFF(SET)
F: 448 745Y 6.2E
20L
Switch to enable display of external
LIGHT-SENSOR readings (in ONposition). When in OFF-position, the PAR-values stored in an
internal list are effective. This list is created via the LIGHT-CAL
function (see next menu point).
7:EXT.LIGHT-S:ON (SET)
F: 448 745Y 6.2E
20L
Automatized routine for determination of
PAR-values of the 12 ACTINIC LIGHT
Intensity settings in a given measuring geometry. These values are
stored in a list, which is effective whenever the EXT.LIGHT8:LIGHT-CALIB:
(SET)
F: 448 745Y 6.2E
20L
18
CHAPTER 7
THE MODE-MENU
SENSOR is OFF (menu point 7). For this determination the LIGHTSENSOR must be fixed instead of the sample in front of the
fiberoptics. When the routine is carried out, the LIGHT
AVERAGING function (menu point 6) is disabled. If it is afterwards
required, it must be manually enabled. After the LIGHTCALIBRATION the EXT.LIGHT-SENSOR (menu point 7) is in the
OFF-position.
When in ON-position, the DISPLAY is
continuously illuminated. It should be
noted, that this may cause considerable costs of battery power. When
in OFF-position, DISPLAY ILLUMINATION can be transiently
turned on for 40 s by pressing ON for 3 s.
9: DISP.ILLUM:OFF(SET)
F: 448 745Y 6.2E
20L
On/off switch to enable/disable the power
saving automatics which turn off the
MINI-PAM after 4 min without key operation. It is advisable to
disable the AUTO-OFF when the MINI-PAM is connected to an
external power supply (via CHARGE-socket). Whenever the
instrument is switched off manually, the AUTO-OFF function is
enabled again (automatic reset to ON-position). The AUTO-OFF
function is also automatically enabled when battery voltage drops
below 11.2 V.
10: AUTO-OFF: ON (SET)
F: 448 745Y 6.2E
20L
Function to average a number of
consecutive YIELD- and ETR-determinations. The SET-key is used to reset the counter to 0 and to erase
the averaged values of the preceding measurements. For safety's sake
the reset must be confirmed by pressing the ∧-key. The averaged
YIELD and ETR are shown in the top line, whereas in the bottom
line the values of the last measurement are displayed.
11:AV. 564Y
F: 448 745Y
5.9E
6.2E
8No
20L
On/off switch of the internal actinic light
source (halogen lamp). This can also be
directly operated via ON + SET. The internal actinic lamp is not
12: ACT-LIGHT: OFF(SET)
F: 448 745Y 6.2E
20L
19
CHAPTER 7
THE MODE-MENU
meant to be turned on for extended periods of time, as this may lead
to excessive internal heating. Therefore, the illumination periods are
restricted (see menu point 14: ACT-WIDTH). There is a blinking
sign (ACT) in the upper left corner while actinic illumination is on.
On/off switch of the internal actinic light
source, with additional application of a
saturation light pulse for YIELD-assessment at the end of the
illumination time which is set by menu point 14: ACT-WIDTH.
There is a blinking sign (A+Y) in the upper left corner of the display
while actinic illumination with terminal YIELD-determination is
running. This function can be also directly started from standard
position 1 by double key operation ON + START.
13: ACT+YIELD: OFF(SET)
F: 448 745Y 6.2E
20L
Setting of actinic illumination time. The
setting can be modified via SET and the
arrow-keys in 10 s steps. Maximal setting is limited to 5 min (5:00)
in order to avoid excessive internal heating.
14:ACT-WIDTH 0:30 (SET)
F: 448 745Y 6.2E
20L
Setting of intensity of internal actinic light
source (halogen lamp). The setting can be
modified via SET and the arrow-keys between 0 and 12. The range
covered by intensities 1-12 can be shifted up and down with the help
of AL-FACT (menu point 16).
15: ACT-INT: 5 (SET)
F: 448 745Y 6.2E
20L
Actinic light factor by which the range of
actinic intensities (ACT-INT, menu point
15) can be shifted up and down. The standard factor of 1.00 can be
modified between 0.5 and 1.5 via SET and the arrow keys. The
relationship between AL-FACT and PAR is non-linear.
16: AL-FACT: 1.00 (SET)
F: 448 745Y 6.2E
20L
When switched on via SET, first the
maximal YIELD in the absence of actinic
light (Fv/Fm) is measured and then a series of 8 consecutive YIELDmeasurements at increasing light intensities is started. This function
17:LIGHT CURVE:OFF(SET)
F: 448 745Y 6.2E
20L
20
CHAPTER 7
THE MODE-MENU
can be also directly started by double key operation ON + ∧. The
time periods at the different intensities are set by menu point 19: LCWIDTH. There is a blinking sign (LC) in the upper left corner of the
display while a LIGHT CURVE is recorded. The series involves
YIELD-determinations at 8 settings of actinic light. It starts with the
intensity-setting, which is selected by 20: LC-INT, where one can
choose between values from ACT-INT 1 to 5, with the standard
setting being ACT-INT 3. The range of absolute PAR-values
corresponding to these settings can be moved up and down with the
help of menu point 16: AL-FACT or by changing the distance
between fiberoptics and sample. The effective PAR-values at the
sample surface may be calibrated by the LIGHT-CALIBRATION
routine (menu point 8). A LIGHT CURVE can provide profound
information on the overall photosynthetic performance of a plant,
even if the illumination periods are too short to achieve true steady
states. Note: Due to the unavoidable internal heating during
recording of a LIGHT CURVE, assessment of absolute fluorescence
signal amplitudes is problematic, but this does not affect correct
determination of the ratio ΔF/Fm'.
When switched on via SET, a LIGHT
CURVE is measured as described for menu
point 17 and in the following dark period the recovery of YIELD is
assessed by 6 consecutive measurements at 10 s, 30 s, 60 s, 2 min,
5 min and 10 min following illumination. Note: Due to the
unavoidable internal heating during recording of a LIGHT CURVE
assessment of absolute fluorescence signal amplitudes is
problematic, but this does not affect correct determination of the ratio
ΔF/Fm'.
18:L.CURVE+REC:OFF(SET)
F: 448 745Y 6.2E
20L
19: LC-WIDTH 0:10 (SET)
F: 448 745Y 6.2E
20L
LC-WIDTH determines the illumination
time at each intensity setting. 10 s are
21
CHAPTER 7
THE MODE-MENU
sufficient for so-called "rapid light curves". It is limited to 3 min in
order to avoid excessive internal heating.
The LC-INT determines the starting
intensity which can be chosen between
settings 1 to 5. LIGHT CURVES always involve 8 intensities. Hence,
more emphasis may be put either on the linear rise or on the plateau
region of the curve.
20:
F: 448
LC-INT: 3 (SET)
745Y 6.2E
20L
This function starts registration of a darkto-light INDUCTION CURVE with
Saturation Pulse Quenching Analysis. Normally dark-adapted
samples are used. First a saturation pulse is given for determination
of Fo, Fm and Fv/Fm. After a certain dark time, set by IND. DELAY
(menu point 23), ACTINIC LIGHT at a given intensity (ACT-INT,
menu point 15) is turned on and 8 saturation pulses are applied at
intervals determined by IND.WIDTH (menu point 24).
21: IND.CURVE: OFF(SET)
F: 448 745Y 6.2E
20L
In addition to the recording of dark-to-light
INDUCTION CURVE (as described for
menu point 21), after turning off the ACT.-LIGHT 6 saturation
pulses are applied at 10 s, 30 s, 60 s, 2 min, 5 min and 10 min to
assess the dark recovery of fluorescence parameters.
22: IND.C+REC: OFF(SET)
F: 448 745Y 6.2E
20L
Delay time between first saturation pulse
and turning-on of ACT-LIGHT. The
default setting is 40 s. Possible settings range from 5 s to 10 min.
23:IND-DELAY 0:40 (SET)
F: 448 745Y 6.2E
20L
Time interval between two consecutive
saturation pulses during recording of
IND.CURVE. The default setting is 20 s. Possible settings range
from 5 s to 3 min.
24:IND-WIDTH 0:20 (SET)
F: 448 745Y 6.2E
20L
25:Fo: 530 Fm:2650(SET)
F: 448 745Y 6.2E
20L
22
Function
to
sample
the
minimal
CHAPTER 7
THE MODE-MENU
fluorescence, Fo, and maximal fluorescence, Fm, of a dark-adapted
sample by use of the SET-key. The thus sampled values are stored
until new values are sampled via SET. With START a normal
YIELD-determination is carried out and the given Fo- and Fm-values
are maintained. The stored Fo- and Fm-values are used for
determination of the quenching coefficients qP, qN and NPQ (see
menu points 26 and 27). In some applications, in order to obtain
minimal Fo it is advantageous to make use of the ML-BURST
function (see menu point 5).
Coefficients of photochemical quenching,
qP, and non-photochemical quenching, qN,
as defined by the following equations:
26: qP:1000qN:000 (SET)
F: 448 745Y 6.2E
20L
qP=(M-F)/(M-Fo) and qN=(Fm-M)/(Fm-Fo)
In order to obtain the usual values between 0 and 1, the displayed
values have to be multiplied by 0.001. qP is set to 000 if M<F and
qN is set to 000 if M>Fm. qN is set to 1.000 if M<Fo.
Note: M here represents the maximal fluorescence measured by a
saturation pulse in any given light state (normally denoted Fm'),
whereas Fm and Fo are the particular values sampled via menu point
25 after dark-adaptation. The thus determined values of qP and qN
should be considered approximations only, as a possible nonphotochemical quenching of Fo is not taken into consideration.
27: NPQ:1.440
(SET)
F: 448 745Y 6.2E
20L
Parameter describing non-photochemical
quenching defined by the equation:
NPQ = (Fm-M)/M
Note: M here represents the maximal fluorescence measured by a
saturation pulse in any given light state (normally denoted Fm'),
whereas Fm is the particular value sampled via menu point 25 after
dark-adaptation. NPQ has been shown to be closely related to the
23
CHAPTER 7
THE MODE-MENU
excess light energy which is actively dissipated by plants into heat in
order to avoid photodamage. Contrary to qN, NPQ-determination
does not require knowledge of Fo and is not affected by nonphotochemical quenching of Fo. NPQ is set to 0.000 if M>Fm.
On/off switch of repetition clock which
serves to trigger a number of functions
which are specified in menu point 29: CLOCK ITEM. This function
can be also directly started by double key operation ON + MEM.
28: REP-CLOCK: OFF(SET)
F: 448 745Y 6.2E
20L
This menu point allows to choose between
the following functions to be triggered by
the REPETITION CLOCK:
29: CLOCK-ITEM:SAT(SET)
F: 448 745Y 6.2E
20L
SAT-PULSE, ACT-LIGHT, ACT + YIELD, LIGHT CURVE,
L-CURVE + REC., IND. CURVE , IND.C + REC.
Setting of clock interval, which is the time
between two consecutive saturation pulses
(or other functions) triggered by the REP-CLOCK (menu point 28).
The setting can be modified via SET and the arrow-keys in 10 s
steps. Possible settings range from 0:10 to 42:30. When moving
beyond the maximal time, the lowest values are reached and vice
versa.
30: CLK-TIME: 0:30(SET)
F: 448 745Y 6.2E
20L
Display of present time which can be
modified via SET and the arrow-keys.
With SET one can move from the hours to minutes and vice versa.
The change is terminated via MODE.
31: TIME 14:43:51 (SET)
F: 448 745Y 6.2E
20L
Display of present date which can be
modified via SET and the arrow-keys.
With SET one can move from the days to months and vice versa. The
change is terminated via MODE.
32: DATE 17-OCT (SET)
F: 448 745Y 6.2E
20L
33: YEAR 1999
(SET)
F: 448 745Y 6.2E
20L
24
Display of present year which can be
CHAPTER 7
THE MODE-MENU
modified via SET and the arrow-keys. The change is terminated via
MODE.
Display of battery voltage. The value in
brackets shows the voltage observed
during the last saturation pulse (transiently decreased value due to
high current of halogen lamp). YIELD-measurements may become
erroneous, if the voltage during a pulse drops below 8.0 V (Error
message 6: CHECK BATTERY). The battery voltage is a non-linear
function of the remaining battery capacity. When dropped below
11.2 V (without saturation pulse) the remaining capacity is approx.
20 % and recharging soon will become necessary. In this case there is
a warning (BAT-sign blinking in the left corner of the upper display
line).
34: BATT: 12.8V (12.3)
F: 448 745Y 6.2E
20L
Display of internal temperature of the
instrument which is measured close to the
optical unit. An increase of temperature causes a decrease of
measuring light intensity and, hence, simulates a decrease of
fluorescence yield. This may effect measurements of qP, qN and
NPQ, but not of YIELD and ETR.
35: INT.TEMP: 24C
F: 448 745Y 6.2E
20L
Command to reset all instrument settings
(which can be varied via the MODEmenu) to the standard settings preset at the factory (see in section
7.1).
36: RES.SETTINGS: (SET)
F: 448 745Y 6.2E
20L
37: PROGR.M2.24(170299)
F: 448 745Y 6.2E
20L
Number and date of origin of current
program version of the MINI-PAM which
is resident on EPROM.
Function to move the present MEMORYfront to any number between 1 and 4000.
This function may be important when the MEMORY is full and the
user wants to avoid overwriting of certain older data.
38: MEMORY: 125 (SET)
F: 448 745Y 6.2E
20L
25
CHAPTER 7
THE MODE-MENU
Note: The MEMORY-front is identical to the MEM-number under
which the last data set was stored. It advances by 1 with each
following YIELD-determination.
Command to erase all data accumulated in
MEMORY. For safety's sake this command
is not yet carried out by SET but requires confirmation by pressing
the ∧-key. Then the MEMORY-front is reset to 0 and the data set
recorded with the next saturation pulse will be in MEM position 1.
39: CLEAR MEMORY (SET)
F: 448 745Y 6.2E
20L
Function for adjustment of PAR-reading
by comparison with calibrated device.
Particular care must be taken that both sensors are exposed to the
same photon flux density. After SET, the PAR-reading (L) can be
adjusted by the arrow-keys in steps of 1 μmol quanta m-2 s-1. For
proper calibration over a wide range of PAR also adjustment of
LIGHT-GAIN (menu point 41) may be required. This can be checked
by comparison with calibrated device at a different PAR-value.
40: LIGHT-OFFS: 20(SET)
F: 448 745Y 6.2E
20L
Function for adjustment of PAR-reading.
The adjustment via LIGHT-GAIN should
be carried out after a preceding adjustment by LIGHT-OFFS (menu
point 40) at a different light intensity, such that the slope of the
response curve can be evaluated. For highest accuracy, the LIGHTOFFS then may have to be adjusted once more (menu point 40).
41:LIGHT-GAIN:1.00(SET)
F: 448 745Y 6.2E
20L
Function for adjustment of leaf
temperature-reading with optional LeafClip Holder 2030-B in comparison with calibrated device. After SET,
the temperature reading can be adjusted by the arrow-keys in 0.1 °C
steps. For proper calibration over a wide temperature range also
adjustment of TEMP-GAIN (menu point 43) may be required. This
can be checked by comparison with a calibrated device at different
temperatures.
42:TEMP.OFFS: 0.0(SET)
F: 448 745Y 6.2E
20L
26
CHAPTER 7
THE MODE-MENU
Function for adjustment of leaf
temperature-reading with optional LeafClip Holder 2030-B. The adjustment via TEMP-GAIN should be
carried out after a preceding adjustment by TEMP-OFFS (menu
point 42) at a different temperature, such that the slope in the
temperature response curve can be evaluated. For highest accurracy,
TEMP-OFFS then may have to be adjusted once more (menu point
42).
43:TEMP.GAIN:1.00 (SET)
F: 448 745Y 6.2E
20L
Display of present zero offset value which
normally is identical to the value obtained
automatically via AUTO-ZERO (menu point 2). Following SET, this
value can be manually modified using the arrow-keys.
44: ZERO-OFFS: 20(SET)
F: 448 745Y 6.2E
20L
Display of current factor applied for
calculation of relative electron transport
rate (ETR) which for a standard leaf is defined as follows:
45: ETR-FAC: 0.84 (SET)
F: 448 745Y 6.2E
20L
ETR = Yield x PAR x 0.5 x 0.84
The standard factor 0.84 corresponds to the fraction of incident light
absorbed by a leaf. The preset value, which corresponds to an
average observed with a variety of leaf species, can be modified via
SET and the arrow-keys.
Setting of the width of saturating light
pulses for YIELD-determination. The
setting can be changed between 0.4 and 3.0 s in 0.2 s steps.
46: SAT-WIDTH:0.8s(SET)
F: 448 745Y 6.2E
20L
Setting of saturation pulse intensity for
YIELD-determination. Settings can be
changed between 0 and 12.
47: SAT-INT: 8 (SET)
F: 448 745Y 6.2E
20L
48: DAMP: 2
(SET)
F: 448 745Y 6.2E
20L
Setting of electronic signal damping. The
three settings correspond to the following
27
CHAPTER 7
THE MODE-MENU
time constants (defined for 63.2 % of a signal change): 1: 0.05 s,
2: 0.2 s, 3: 1 s.
Setting of electronic signal gain
(amplification factor) which can be varied
between 1 and 12. By increasing GAIN not only the signal but also
the noise increases in proportion. Any change in GAIN requires a
new determination of the unavoidable background signal via AUTOZERO (menu point 2).
49: OUT-GAIN: 2 (SET)
F: 448 745Y 6.2E
20L
Setting of intensity of measuring light
which can be varied between 0 and 12.
Any change in MEAS-INT requires a new determination of the
unavoidable background signal via AUTO-ZERO (menu point 2).
50: MEAS-INT: 8 (SET)
F: 448 745Y 6.2E
20L
Letter from A to Z for identification of a
particular type of sample. This MARK is
entered into the MEMORY with every new data set measured in
connection with a saturation pulse. It can be helpful when a number
of different plants are assessed in the field.
51: MARK: A
(SET)
F: 448 745Y 6.2E
20L
28
CHAPTER 8
COMPONENTS OF THE MINI-PAM
8 Components of the MINI-PAM
The basic functional system for measurements of fluorescence
yield and of the effective yield of photosynthetic energy conversion
consists of the MINI-PAM Main Control Unit and the fiberoptics.
Additional peripheral components can be connected to the four
sockets at the rear side of the Main Control Unit. Fig. 2 shows a
functional block diagram of the MINI-PAM and its most essential
accessories.
Rechargeable battery
Power saving technology
CHARGE
Battery Charger
MINI-PAM/L
External battery
LC display
Tactile key pad
control
Central Processing Unit
Selective amplifier
AD converter
Data storage/analysis
OUTPUT
analog
MINI-PAM
Computer control
RS 232
Data analysis
WinControl
Software
Data transfer
LEAF CLIP
PAR, °C
Modulated measuring light
Saturation pulse light
Actinic light
Data storage/analysis
Chart recorder
MINI-PAM/F
Fiberoptics
Printer
Remote
control
Leaf Clip
Holder 2030-B
External Halogen
Lamp 2050-HB
Main Control Unit
Fig. 2
8.1
Main Control Unit
Except for the fiberoptics, which are attached to it, the Main
Control Unit contains all essential components of the MINI-PAM
29
CHAPTER 8
COMPONENTS OF THE MINI-PAM
Fluorometer. These include the optics for fluorescence excitation and
detection, the selective amplifier, the data acquisition and storage
system, an actinic light source for saturation pulses and continuous
illumination, a large rechargeable battery and the user interface, with
the LC-display and keyboard. Details on some of these components
are given in the following sections.
8.1.1
Fluorescence excitation and detection
In the standard version of the MINI-PAM fluorescence is excited
by pulse modulated red light from a light-emitting-diode (LED). The
pulse-width is 3 μs and pulse frequency is 0.6 or 20 kHz. In the socalled "burst-mode" pulse trains of 0.2 s are alternating with 0.8 s
dark-intervals. The LED-light is passed through a cut-off filter
(Balzers DT Cyan, special) resulting in an excitation band peaking at
650 nm, with a very small "tail" at wavelengths beyond 700 nm.
Fluorescence is detected with a PIN-photodiode at wavelengths
beyond 700 nm, as defined by a long-pass filter (type RG 9, Schott).
The effective intensity of the measuring light at the level of the
sample is an important parameter for correct determination of the
minimal fluorescence yield, Fo, of a dark-adapted sample. Its
absolute value depends on
• intensity setting (menu point 50, preset value 8),
• measuring frequency (menu point 4, preset at 0.6 kHz),
• burst mode status (menu point 5, preset to be off),
• distance between fiberoptics and sample (standard 12 mm).
At the standard distance of 12 mm between fiberoptics and
sample, and at measuring light intensity 8, the quantum flux density
of photosynthetic active radiation typically amounts to 0.15 μmol
30
CHAPTER 8
COMPONENTS OF THE MINI-PAM
quanta m-2 s-1 at 0.6 kHz and 5 μmol quanta m-2 s-1 at 20 kHz. These
values are lowered to 1/5 when the burst mode is active. At such low
intensities an "actinic effect" of the measuring light normally can be
excluded.
8.1.2
Special information on MINI-PAM/B
Recently strong blue LEDs with an emission peak around 470
nm have become available and the MINI-PAM/B was developed
which employs such LED as measuring light source. Using 470 nm
measuring light has a number of technical and practical
consequences which shall be briefly outlined.
•
Excitation filters:
The 470 nm LED light is passing through a set of short-pass
filters with λ < 620 nm (Balzers DT Cyan special).
•
Detector filters:
The PIN-photodetector is protected by a set of long-pass filters
with λ > 650 nm (Balzers R65 and Schott RG 645).
•
Measuring light:
At the same intensity setting (MEAS-INT) the integrated
intensity of the measuring light pulses of the blue version is less
than that of the red version by approximately a factor of 3.
Therefore, the MEAS-INT can be applied at higher settings
without the risk of an actinic effect.
•
Actinic light:
The actinic light passes the same filters as the measuring light.
Therefore, the short-pass wavelength of 620 nm not only applies
to measuring light but to actinic light and saturation pulse light
as well. Consequently, as the red component of the halogen lamp
31
CHAPTER 8
COMPONENTS OF THE MINI-PAM
is cut off, the absolute values of actinic light intensities are lower
than in the standard instrument version and in order to obtain
equivalent PAR-values, correspondingly higher intensity settings
must be chosen.
•
Spectral shifts:
It is an unavoidable property of halogen light sources that the
emission spectrum shifts from red to blue when the light output
increases with increasing power. This property may complicate
the assessment of photosynthetically active radiation and
consequently of relative electron transport rate (ETR) as well. As
the red part of the spectrum is not used in the MINI-PAM/B, this
aspect is less problematic than in the standard instrument
version.
•
Chlorophyll excitation:
In most photosynthetic organisms blue light excites chlorophyll
fluorescence about as well as red light. However, in organisms
with phycobilisomes (cyanobacteria and red algae) the yield of
blue light excited fluorescence is rather low. This is due to the
fact that most of the chlorophyll in these organisms is associated
with photosystem I and in a low-fluorescent state. Therefore, the
use of the blue instrument versions cannot be recommended for
the study of such organisms (e.g. also lichen with cyanobacteria
as photobionts).
32
CHAPTER 8
•
COMPONENTS OF THE MINI-PAM
Chlorophyll emission:
As the cut-off wavelength of the detector filter is shifted from
710 nm in the standard version to 650 nm in the blue version, the
latter also detects the main chlorophyll emission peaking around
685 nm which originates mainly from photosystem II and, hence,
shows higher values of variable fluorescence.
8.1.3
Internal halogen lamp as actinic light source
A miniature 8 V/20 W halogen lamp (type Bellaphot, Osram)
serves as light source for saturation pulses and for continuous actinic
illumination. The light is filtered two-fold by a heat-reflecting filter
(Balzers, Calflex-X, special) and by a short-pass filter (Balzers, DT
Cyan, special), such that white light with negligible content of
wavelengths beyond 700 nm (standard version) or beyond 640 nm
(MINI-PAM/B) is obtained.
It is not recommended to operate the internal halogen lamp for
extended periods of actinic illuminations as this would lead to
excessive internal heating. This aspect must be taken particularly
serious when light curves are automatically recorded (menu points
17 and 18) and when the range of actinic intensities is increased by
AL-FACT (menu point 16). A temperature-sensor, which is mounted
in the vicinity of the lamp, causes turn-off of the lamp power supply
when 70 °C is reached. It is turned on again when temperature has
dropped to approx. 55 °C. The internal temperature, the value of
which is displayed under menu point 35, affects the output of the
measuring light LED. A 1 °C temperature rise leads to approx. 1 %
lowering of the measuring light intensity. While not affecting the
actual YIELD-measurement (i.e. ΔF/Fm'), this will lead to a
corresponding drop in the fluorescence signal. For prolonged actinic
illumination, particularly at high intensities, the External Halogen
33
CHAPTER 8
COMPONENTS OF THE MINI-PAM
Lamp 2050-HB in combination with the Leaf-Clip Holder 2030-B is
recommended.
8.1.4
Rechargeable battery
A relatively large rechargeable lead acid battery (12 V/2 Ah) is
mounted in the bottom of the MINI-PAM housing. For recharging,
the Battery Charger MINI-PAM/L is provided which is connected to
the CHARGE-socket at the rear side of the MINI-PAM. The charger,
which operates at input voltages between 100 and 240 V AC,
features an overload protection. Full charging of an empty battery
takes approx. 5 hours. Battery voltage is displayed under menu point
34. The warning 'BAT' is given in the upper left corner of the display
when voltage drops below 11.2 V in the resting state. If in this
situation the AUTO-OFF function (menu point 10) is disabled, it will
be automatically enabled again. In addition, there is the warning Err.
3: 'LOW BATTERY' which, however, is coupled to measurements
involving START. After this error message approximately 20 further
measurements can be made and the battery should be soon recharged.
In brackets also the voltage is given which was measured during the
last saturation pulse. It is normal that voltage drops by 0.5 V during a
saturation pulse. However, if it drops below 8 V, YIELDmeasurements may become erronous, as Fm' is likely to be
underestimated. In this case, there is the warning Err. 6: 'CHECK
BATTERY'.
With a fully charged battery the displayed voltage is 12.5 - 12.9
V. In first approximation, battery voltage can be taken as a measure
of remaining battery power. The functional relationship between
capacity (Ah) and voltage of a new battery is depicted in Fig. 3. It is
apparent that battery voltage first drops steeply to about 12.3 V and
then slowly decreases to about 11.8 V, from whereon there is a steep
further drop to values below 11 V.
34
CHAPTER 8
COMPONENTS OF THE MINI-PAM
The MINI-PAM can be also powered by an external 12 V battery
for which purpose a special cable (MINI-PAM/AK) is available
which can be connected to the CHARGE-socket at the rear side of
the MINI-PAM. It should be noted, that a recharging of the internal
battery with a 12 V external battery is not possible.
Fig. 3
8.1.5
LC-display
The data are displayed by a 24 x 2 character LC-display with
backlight. The backlight, which switches on together with the
instrument, automatically turns off again after 50 s, for the sake of
saving battery power. It can be turned on again for 50 s by pressing
the ON-key for at least 3 s. Display illumination can also be switched
on permanently via menu point 9: DISP.ILLUM. However,
permanent backlight operation is recommended only when the
35
CHAPTER 8
COMPONENTS OF THE MINI-PAM
battery charger is connected, as it increases basic power consumption
from 0.7 W to 1.5 W.
The information shown on the LC-display is intentionally
restricted to the most relevant parameters. Additional information can
be called on display in connection with the 51 menu points and by
entering the MEMORY.
8.1.6
Electronic components
The extremely compact design of the MINI-PAM is a
consequence of recent progress in miniaturization of solid state
integrated circuits. The central processing unit features a powerful
CMOS microcontroller. The program software is stored in a CMOS
EPROM. This EPROM is readily accessible after removing the
bottom of the MINI-PAM (see 11.4) and can be exchanged by the
user, if program up-dates become available. A CMOS RAM with 128
kB serves as data memory, providing storage capacity for 4000 data
sets.
36
CHAPTER 8
8.1.7
COMPONENTS OF THE MINI-PAM
Description of the connectors
LEAF CLIP
RS 232
OUTPUT
CHARGE
Fig. 4
At the rear side of the MINI-PAM besides the optical fiber
connector the following electrical connectors are located:
a) LEAF-CLIP
The LEAF CLIP socket can be used for connecting the Leaf-Clip
Holder 2030-B (with integrated micro-quantum-sensor and
temperature-sensor) or the separate Micro-Quantum/Temp.-Sensor
2060-M. Connection of one of these devices is required for display
of L (light intensity, given in quantum flux density of
photosynthetically active radiation, PAR, i.e. µmol quanta m-2 s-1), E
(apparent electron transport rate, ETR) and C (leaf temperature in
°C). See also section 8.3.
b) RS 232
An RS 232 interface cable is provided to connect the MINI-PAM
to IBM or IBM-compatible PCs for operation under WinControl
37
CHAPTER 8
COMPONENTS OF THE MINI-PAM
software, data transfer (see 9) or for remote control of the MINIPAM functions via PC keyboard operation (see 13.4).
c) OUTPUT
A special cable is provided to connect the MINI-PAM analog
output to a chart recorder. The output signal can vary between 0 and
4 volt.
d) CHARGE
Together with the MINI-PAM the Battery Charger MINI-PAM/L
is delivered which connects to the CHARGE-input at the rear side of
the instrument. The charger can be used with line voltages of 100 to
240 V at 50-60 Hz. When used in the laboratory the charger can
remain permanently connected. A special cable (MINI-PAM/AK) is
available for connecting an external 12 V battery to the CHARGEinput. While the MINI-PAM can be powered by this external battery,
it should be noted that the internal battery cannot be recharged in this
way.
8.2
Fiberoptics MINI-PAM/F and Miniature Fiberoptics
MINI-PAM/F1
The fiberoptics are inserted into the corresponding adapter at the
rear side of the MINI-PAM. The active cross section of the standard
version MINI-PAM/F is 5.5 mm. A special version (MINI-PAM/F1)
with Ø 2 mm is also available, consisting of a single plastic fiber. In
the standard version, numerous 70 µm fibers are thoroughly
randomized over a 100 cm mixing pathway, such that a homogenous
field of illumination is created. A so-called 'Distance Clip' (see Fig.
5) is provided with the fiberoptics for convenient positioning of the
fiberoptics end-piece relative to the sample.
38
CHAPTER 8
COMPONENTS OF THE MINI-PAM
Fig. 5
Two spacer rings may be used to define fixed distances. The
fiberoptics exit plane is positioned at a 60° angle relative to the
sample plane. In this way shading of the sample is minimized, if the
fiberoptics are pointing towards the sample from the side opposite to
incident light. The sample may be placed either below the hole or,
preferentially with normal leaves, above the hole. In the latter case,
the leaf can be held between the folded part of the clip. The former
possibility applies e.g. to thick leaves, lichens and mosses. The
distance between fiberoptics exit plane and sample has considerable
influence on signal amplitude and effective light intensities.
Unavoidably, with a 60° angle between sample plane and fiberoptics
there is a range of distances between fiberoptics and leaf which will
result in a light intensity gradient. The relative magnitude of this
gradient is reduced with increasing fiber distance. However, this
point should not be of too much concern, as there is anyway a larger
vertical light gradient within the leaf due to chloroplast shading by
the top chloroplast layer. Also, the measured signal will be
dominated by that part or the leaf which receives maximal intensity,
39
CHAPTER 8
COMPONENTS OF THE MINI-PAM
as this also is most strongly excited by the measuring light and emits
most of the fluorescence which is received by the fiberoptics.
For measurements with leaves the special Leaf-Clip Holder
2030-B was developed, featuring an integrated micro-quantumsensor and a thermocouple (see 8.3). For this holder also a 90°
fiberoptics adapter (2030-B90) is available.
The fiberoptics should be handled with care. Excessive bending,
in particular close to the connector plug, should be avoided, as it
would lead to fiber breakage with resulting loss in signal amplitude.
The fibers are protected by a steel-spiral and plastic mantle which
provides a natural resistance to strong bending.
In addition to the standard fiberoptics MINI-PAM/F, with an
active diameter of 5.5 mm, a miniature fiberoptics with 2 mm active
diameter (MINI-PAM/F1) is available for small spot measurements.
This is particularly recommended for use in conjunction with the
Portable Photosynthesis System HCM-1000. For this purpose a
modified top window of the HCM-1000 can be provided which
allows to approach the clear fibertip at 60° angle close to the leaf
surface with minimal shading of the sample. Combined
measurements of fluorescence and gas exchange provide unique
complementary information on the photosynthetic performance of a
plant.
Using the Miniature Fiberoptics MINI-PAM/F1 the signal
amplitude is particularly sensitive to the distance between fibertip
and sample. A standard distance of 4 mm provides for a homogenous
field of illumination and a very satisfactory signal amplitude,
approximately equal to that obtained with the 5.5 mm Ø fiberoptics
at the standard distance of 12 mm. Signal amplitude can be further
increased at least 4 fold, when the fibertip is advanced to the sample
surface. It should be noted, however, that in this case the measuring
40
CHAPTER 8
COMPONENTS OF THE MINI-PAM
light may show an actinic effect. This can be counteracted by use of
the ML-BURST function (MODE-menu position 5, see 12.3.2).
Because of the strong influence of sample distance on signal
amplitude, particularly with the 2 mm Ø fiberoptics, it is
recommended to clamp the fiberoptics tip at fixed distance to the
sample surface. Otherwise there may be substantial errors, even in
the ratio measurement of ΔF/Fm, when the distance changes between
the consecutive measurements of F and Fm, separated by
approximately 1 s (see 12.3.7).
8.3
Leaf-Clip Holder 2030-B
The Leaf-Clip Holder 2030-B may substitute for the standard
'Distance Clip' as a device for defined positioning of the fiberoptics
relative to the leaf plane. The leaf is resting on a perspex tube with
widened crest, which can be vertically adjusted, to account for
different leaf thicknesses. The fiberoptics axis forms a 60° angle with
the leaf plane. In this way shading of the sample can be largely
avoided when external actinic illumination is applied. For special
applications using the internal actinic lamp a 90° fiberoptics adapter
(2030-B90) is available which can be readily mounted instead of the
standard 60° adapter. This is e.g. particular useful for recordings of
LIGHT CURVES, providing more homogenous illumination and
higher signal amplitudes (see 12.3.7). The distance between
fiberoptics and leaf can be varied. Standard distances are defined by
spacer rings. In addition, the Leaf-Clip Holder 2030-B displays the
following features:
41
CHAPTER 8
-
COMPONENTS OF THE MINI-PAM
Micro-quantum-sensor monitoring PAR
This tiny sensor is unique in monitoring the photosynthetic active
radiation (PAR) at the very spot where also fluorescence is measured
and at which photosynthetic performance is assessed. This function
already is fulfilled, when approximately 10 % of the total measuring
area is occupied by the sensor. The resulting loss in signal amplitude
is small. If wished, the sensor can also be moved out of the
measuring field which is limited by a metal ring of 10 mm inner
diameter. With its tip resting on this ring, even without penetrating
into the measuring field the sensor will accurately monitor incident
light intensity under natural day light conditions, when the leaf-clip
holder is positioned such that light incidence is mainly from the
front.
Essential opto-electronical elements of this micro-quantumsensor are a 1.5 mm cross-section diffusing disk; a 0.5 mm diameter
fiber guiding the scattered light to the detector; a filter combination
selecting the photosynthetic active wavelength range between 380
and 710 nm; and a blue-enhanced silicon photodiode. Despite its
small dimensions, the diffuser displays properties of 'cosine
correction', i. e. also light impinging at rather small incidence angles
(e. g. with rising or setting sun) is reliably monitored. Due to the
equalization of leaf and sensor planes, automatically achieved by
fastening the leaf in the clip, the measured effective PAR very
closely corresponds to the PAR at that spot of the leaf where
fluorescence is measured. The micro-quantum-sensor measures
incident photosynthetic radiation in µmol quanta m-2s-1, i.e. in units
of flux density. Hence, the measured parameter is identical to PPFD
(photosynthetic photon flux density). It is displayed at the end of the
second line of the LCD (...L) when the leaf-clip holder is connected.
The sensor was calibrated against a LI-COR Quantum Sensor (Type
LI-190). The stability of calibration depends strongly on keeping the
42
CHAPTER 8
COMPONENTS OF THE MINI-PAM
diffuser clean. Also, it must be pointed out that there is some
decrease in sensitivity when the sensor is moved from the center of
the measuring field to its periphery. It is advisable to check
calibration regularly by comparison with a standard quantum sensor,
like the LI-190. Any deviation can be corrected by entering a
recalibration factor via menu point 41: LIGHT GAIN. A substantial
increase of the calibration factor from its original value of 1.00
indicates dirt-deposition on the diffuser, which may be reversed by
gentle cleaning using a cotton-tip, moistened with some alcohol. In
addition, it is possible to enter an offset value via menu point 40:
LIGHT-OFFS.
-
Thermocouple monitoring leaf temperature
A NiCr-Ni thermocouple is mounted in the perspex tube on
which the studied leaf area is resting. Its tip is forming a loop which
gently presses against the lower surface of the leaf. In this way there
is effective temperature equilibration and the thermocouple is
protected from direct sun radiation. The reference couple is located
on the circuit board, in close proximity to the thermovoltage
amplifier (AD), enclosed in the bottom part of the holder. The
relationship between thermovoltage and temperature is almost linear.
With decreasing temperatures there is a small decline of ΔV/°C.
Calibration was performed at 25 °C. At 0 °C or -15 °C the deviation
amounts to 0.5 or 0.8 °C, respectively. An offset value can be entered
via menu point 42: TEMP-OFFS. The measured temperature is
displayed at the end of the first line of the LCD (...C) when the LeafClip Holder 2030-B is connected. Temperature resolution is 0.3 °C.
The temperature as well as the PAR data are automatically stored in
the memory after every saturation pulse, together with the
fluorescence data.
43
CHAPTER 8
-
COMPONENTS OF THE MINI-PAM
Remote control push button
Pressing the 'remote' control push button on the handle of the
Leaf-Clip Holder 2030-B is equivalent to operation of START on the
MINI-PAM keyboard. In practice, this offers the advantage, that the
experimenter can use both hands for positioning the leaf within the
holder and at the same time trigger a recording by remote control. In
this way, sampling is considerably facilitated, which is particularly
helpful when many recordings are averaged to increase the accuracy
of determinations.
Approx. 0.2 seconds elapse between pushing the remote control
button and triggering of the saturation pulse. The actual start of the
measurement is announced by a beep-sound. From that moment
onward the leaf clip should be held steady for approx. one second.
-
Tripod mounting thread
Mounting the Leaf-Clip Holder 2030-B on a tripod (e. g.
Compact Tripod ST-2101A) facilitates long term recordings with the
same plant. Such recordings can be automated by using the Clockfunction.
-
Holes for mounting External Halogen Lamp 2050-HB
Two holes are provided in the front bottom part of the holder for
mounting the optional External Halogen Lamp 2050-HB (see 8.5).
This lamp allows long periods of illumination with strong light, as e.
g. required for photoinhibitory treatment. It is not recommended to
use the internal halogen lamp for this purpose, as this would lead to
excessive internal heating and rapid depletion of battery power.
44
CHAPTER 8
8.4
COMPONENTS OF THE MINI-PAM
Micro Quantum/Temp.-Sensor 2060-M
The Micro Quantum/Temp.-Sensor 2060-M essentially displays
the same features as outlined above for the Leaf-Clip Holder 2030-B
(see 8.3), except that the micro-sensors of PAR and temperature are
not mounted in a leaf-clip. This device is rather designed for
experiments with objects which are not leaf-shaped, like crustose
lichens and cushions of moss. The two miniature sensors can be
attached to the site where fluorescence is monitored without
interfering with the actual measurement. A defined position with
respect to the object and the fiberoptics exit plane can be achieved
with the help of a special holder, in analogy to the 'Distance Clip'
(shown in Fig. 5).
It should be pointed out that the sensitivity of the micro quantum
sensor is affected by bending the relatively long, flexible light guide
which bridges the distance between the small diffusing disk at the
object and the detector in the metal housing. Therefore, this device
cannot substitute for a reliable quantum sensor like the LI-COR
Quantum Sensor (Type LI-190), against which it was originally
calibrated. Recalibration via menu points 40 and 41 is recommended
when bringing the sensor and the metal housing into a fixed position
with respect to the object.
8.5
External Halogen Lamp 2050-HB
The External Halogen Lamp 2050-HB provides a strong light
source for prolonged illumination periods, for which purpose the
internal halogen lamp is not suited because of the heat developing
within the MINI-PAM housing. A 20 W lamp is powered by an
external battery (e. g. NP-3/12). Its intensity can be varied steplessly
via a 15-turn potentiometer. Power consumption is minimized by
special electronic circuitry. The lamp is equipped with a heat45
CHAPTER 8
COMPONENTS OF THE MINI-PAM
reflecting, sealed window. In addition, for standard applications a
short-pass filter (λ<700 nm) is provided, which is mounted directly
on the lamp. This filter passes almost all visible light and only
eliminates the long wavelength radiation, against which the
fluorescence detector is not protected. For special applications, other
filters (e. g. daylight or blue) are available with which, however, the
maximal possible intensities are lower.
The External Halogen Lamp 2050-HB is meant to be used in
conjunction with PAR-measurements, as performed with the LeafClip Holder 2030-B. In its normal application, it is mounted on the
Leaf-Clip Holder, with the light (8° beam divergence angle) shining
at an approx. 60° incident angle with respect to the leaf plane on the
site where fluorescence and PAR are measured. The optimum angle,
giving maximal PAR and minimal shading by the fiberoptics can be
manually adjusted, preferentially using a white piece of paper instead
of a leaf. With the 15-turn potentiometer defined PAR-values can be
chosen, which are read off the MINI-PAM LC-display. A switch is
provided to turn the lamp on/off.
A major application of the External Halogen Lamp 2050-HB is
the adjustment of defined light intensities for measurements of light
saturation curves under field conditions. For this purpose, the light
obtained from this lamp may substitute or complement the natural
daylight. Intensities corresponding to PAR-values of more than 3000
µmol quanta m-2s-1 can be achieved, exceeding the intensity of direct
sun light. Hence, this light source can be also useful for
photoinhibitory treatment of leaves and of other photosynthesising
organisms in the field. It should be noted that application of such
high light intensities will cause a substantial rise of leaf-temperature,
which is monitored by the thermosensor integrated in the Leaf-Clip
Holder 2030-B and can be read off the LC-display of the MINIPAM.
46
CHAPTER 8
8.6
COMPONENTS OF THE MINI-PAM
Dark Leaf Clip DLC-8
The Dark Leaf Clip DLC-8 weighs approx. 4 g and, hence, can
be attached to most types of leaves without any detrimental effects. It
is equipped with a miniature sliding shutter which prevents light
access to the leaf during a dark-adaptation period and which is
opened for the actual measurement only, when exposure to external
light is prevented by the fiberoptics. Proper dark-adaptation is
essential for determination of the maximal quantum yield Fv/Fm (see
12.3.1).
Different from the other leaf clips, with the Dark Leaf Clip
DLC-8 the fiberoptics are positioned at right angle with respect to
the leaf surface at the relatively short distance of 7 mm. As a
consequence, signal amplitudes are approx. 2-3 times higher than
with the Leaf-Clip Holder 2030-B. In order to avoid signal
saturation, the settings of MEAS-INT (menu point 50) and GAIN
(menu point 49) have to be correspondingly lowered with respect to
the standard settings. For optimal results the burst mode of
measuring light (menu position 5: ML-BURST) is recommended
(see 12.3.2).
When the shutter is still closed and measuring light is on, an
artifactual signal is observed, which is due to a small fraction of the
measuring light which after reflection from the closed shutter
penetrates to the photodetector. However, the reflection is much
smaller when the shutter is opened and the measuring light hits the
strongly absorbing leaf instead of the shiny metal. Therefore, it is
recommended to carry out compensation of the unavoidable
background signal by AUTO-ZERO (menu point 2) with the
fiberoptics end directed into the air.
47
CHAPTER 9
DATA TRANSFER
9 Data Transfer
Since the introduction of the WinControl software, the normal
way of data transfer from the MINI-PAM to a PC is via a special
routine provided by WinControl (see separate manual). In addition,
two other programs are provided for the transfer of data from the
MINI-PAM via RS-232 interface cable to a PC. MS-DOS and
WINDOWS versions of the MINI-PAM DATA TRANSFER program
PAMTRANS are available on the disk which is delivered together
with the MINI-PAM. They are installed as follows:
MS-DOS version: Enter 'A:' and enter 'INSTALL'. After
connection of the RS 232 cable and definition of the communication
port (COM 1, 2, ...) the system is ready for data transfer.
WINDOWS version: Enter 'A:\SETUP' at the Program Manager
level of WINDOWS (select first 'File' and then 'Run'). Then SETUP
will be initialized and PAMTRANS.WIN installed. Before data
transfer can be carried out, the RS 232 cable has to be connected and
the communication port (COM 1, 2, ...) has to be defined.
With the WINDOWS-version transfer of 1000 data sets takes ca.
90 s, as compared to 9 min with the MS-DOS version. The steps
required for carrying out the transfer of defined data sets, which are
almost identical for the two versions, shall be briefly described. Fig.
6 and Fig. 7 illustrate the screen layouts used with MS-DOS- and
WINDOWS-versions, respectively. After start of the program the last
measured data set is entered by default into the 'Last Data' field. The
user can enter MEMORY-numbers defining the limits of the transfer
into the 'First Data' and 'Last Data' fields. Before starting the transfer,
a Destination File must be entered into the corresponding parameter
field. The data are processed as text file and the extension .TXT will
be automatically added to the Destination File name.
48
CHAPTER 9
DATA TRANSFER
Upon start of data transfer (F6 in DOS and START button in
WINDOWS) the data sets will be transferred starting with 'First
Data'. Transfer can be stopped (Esc in DOS and Exit button in
WINDOWS). If the address of 'Last Data' has a lower number than
that of 'Fist Data', after MEM 4000 the transfer continues from
MEM1 upwards.
Update versions of the MINI-PAM Data Transfer Program can be
downloaded from our website http://www.walz.com. In this case,
relevant information concerning the update will be contained in a
Read-me file.
╔══════════════════════════════════════════════════════════════════╗
║ MINI-PAM Data Transfer for MS-DOS V1.1
║
╠═════════════════╦════════════════════════════════════════════════╣
║ First Data:1
:║ Data:
:║
╠═════════════════╬════════════════════════════════════════════════╣
║ Last Data :3
:║ Data:
3, A ,14:23:51, 09/04/96,
66,
64,:║
╠═════════════════╩════════════════════════════════════════════════╣
║ Dest. File:C:\TEST
║
╠══════════════════════════════════════════════════════════════════╣
║ + -> Spin one data set up
║
║ - -> Spin one data set down
║
║ # -> Show selected data set
║
║ F6 -> Start transfer from PAM to destination file
║
║ F10-> Leave MINI-PAM Data Transfer
║
║ Esc-> Abort running transfer
║
╠══════════════════════════════════════════════════════════════════╣
║ First data set to be transferred. Select usig 0-9,+,-,#,Backspace║
║ keys. Return jumps to first parameter.
║
╚══════════════════════════════════════════════════════════════════╝
Fig. 6: MS-DOS version of software PAMTRANS
49
CHAPTER 9
DATA TRANSFER
Fig. 7: WINDOWS version of software PAMTRANS
50
CHAPTER 10 MINI-PAM OPERATION VIA PC-TERMINAL
10 Operation of the MINI-PAM via a PC-Terminal
and the RS 232 Interface
The MINI-PAM is basically conceived as a stand-alone
instrument, i.e. the most essential measurements can be carried out
without the need for any peripheral instruments. However, when
used under laboratory conditions, operation via PC and the dedicated
WinControl software has distinct advantages, particularly with
respect to data display (see separate WinControl manual). In
addition, there is the possibility to connect the MINI-PAM via the RS
232 interface to a PC and to control all functions by PC-keyboard
operations using user-written software. For this purpose, first a
suitable terminal program must be installed which allows
communication between the PC and the MINI-PAM. In WINDOWS
the TERMINAL program is available (under Accessories in the
Program Manager). In order to enable this program for
communication with the MINI-PAM the following steps are required:
• Define communication parameters (SETTINGS-menu)
Baud rate
9600
Data bits
8
Stopbits
1
Parity
none
Protocol
Xon/Xoff
Connector
Com 1 or 2
• Define terminal preferences (SETTINGS menu)
Colums
132
Terminal fond
Fixed sys
• Create MINI-PAM.trm (SAVE AS ... in FILE-menu)
For the communication between PC and MINI-PAM a special set
of commands is provided which is listed in the Appendix (section
13.4). Each command consists of one or several low-case letters
51
CHAPTER 10
MINI-PAM OPERATION VIA PC-TERMINAL
which may be followed by further specifications. Any command is
executed via 'Return'.
The following examples may serve to illustrate the principle of
MINI-PAM TERMINAL operation:
• Enter 'bp' and the MINI-PAM will answer with a beep after
'Return' (which is always required and from here onward will not
be mentioned anymore).
• 'a1' will switch on actinic light, which will be switched off again
by 'a0'.
• 's' starts a saturation pulse
• with 'f' and 'fmp' the values of the fluorescence parameters F and
Fm', as measured with the last saturation pulse, can be called on
display.
In this way, it is possible to carry out all MINI-PAM functions by
remote control from a PC terminal and to transfer information from
the MINI-PAM to a PC. In principle, using the TERMINAL-program
also a network of MINI-PAM Fluorometers can be operated.
52
CHAPTER 11
MAINTENANCE
11 Maintenance
11.1 Internal battery and its replacement
The internal battery is essentially 'maintenance free'. However,
even when the instrument is switched off, there is some discharge,
which is stimulated by elevated temperatures. If it is forseeable that
the instrument will not be used for some months, the battery should
be charged beforehand. Excessive discharge of the battery should be
avoided, as this may cause irreversible damage. Such damage
involves lowering of the capacity and increase of internal resistance,
with the consequence that recharging becomes necessary after
relatively short times of operation and that there is an excessive
lowering of voltage during a saturation pulse. In this case, battery
replacement is recommended.
The MINI-PAM features a number of functions and warnings
which make it highly unlikely that excessive discharge of the battery
occurs inadvertently:
• AUTO-OFF (when there was no key operation for 4 min)
• Backlight-off (50 s after switching the instrument on)
• Menu point 34: BATT (display of battery voltage in the resting
state as well as with application of a saturation pulse)
• Warning 'BAT' on the display, when battery voltage drops below
11.2 V in the resting state
• Error message 3: 'LOW BATTERY' when battery voltage drops
below 11.2 V (coupled to measurements involving START).
• Error message 6: 'CHECK BATTERY' when battery voltage
drops below 8.0 V during a saturation pulse.
53
CHAPTER 11
MAINTENANCE
• When battery voltage drops below 8.0 V the CLOCK is
automatically turned off. This is important as the CLOCK
disables the AUTO-OFF function.
If replacement of the battery becomes necessary, this is readily
accessible after removing the 4 screws at the bottom of the MINIPAM. The battery is attached to the bottom piece by double-sided
adhesive tape. After disconnecting the cables, the battery can be
detached by means of a screw-driver used as a lever. The
replacement battery comes with adhesive tape. When connecting the
cables, please note the proper contact polarities (red/positive and
black/negative).
11.2 Halogen lamp and its replacement
Due to a very efficient optical system, very high light intensities
can be obtained with the internal 8 V/20 W halogen lamp, without
applying the maximal allowable voltage. This results in a long life
time of the lamp which is primarily meant to generate saturation
pulses. Continuous operation is limited to 5 min periods in order to
avoid excessive internal heating of the MINI-PAM. For longer
illumination periods we recommend the External Halogen Lamp
2050-HB in combination with the Leaf Clip Holder 2030-B.
For replacement of the internal halogen lamp the MINI-PAM is
opened by removing the bottom part (4 screws). The lamp is held in
a pre-focused position by an aluminum mounting-frame, which is
fastened to the optical compartment by two screws. These screws can
be removed with a hex nut screw driver delivered together with the
MINI-PAM. Spare Halogen Lamps (SL-8/20) with mounting-frame
are available.
54
CHAPTER 11
MAINTENANCE
11.3 Fuse replacement
Two fuses are provided:
4 A: halogen lamp circuit
500 mA: general electronics
For replacement, put the MINI-PAM upside down and remove
the bottom part (4 screws). The fuses are located on the main board,
with the 500 mA fuse being more close to the side featuring the
various connectors.
11.4 EPROM and its replacement
The location of the EPROM on the microcontroller board is
indicated in Fig. 8, which shows a view on the interior of the MINIPAM in its upside-down position after removing the bottom part (4
screws). The EPROM contains the software of the current program
version (see menu point 37). It can be readily exchanged against a
new EPROM when program updates become available. Please note
the little red dot at the side of the EPROM which is directed to the
side of the MINI-PAM housing where the various connectors are. For
lifting the EPROM, a paper-clip can be useful. Put a finger on it, so
that it does not jump up. When installing the replacement, make sure
that the red dot is on the proper side (there is also an arrow on the
EPROM socket). Push in the EPROM firmly, until there is a click
and it sits level at all sides. After EPROM replacement it is
recommended to reset the instrument settings and to clear the
memory (menu point 36 and 39).
55
CHAPTER 11
Fig. 8
56
MAINTENANCE
CHAPTER 12
MEASUREMENTS WITH THE MINI-PAM
12 Chlorophyll Fluorescence Measurements
with the MINI-PAM
Chlorophyll fluorescence is a large signal and in principle its
measurement is rather simple. Hans Kautsky already observed
chlorophyll fluorescence changes by his bare eyes in 1931 and
suggested that these are related to photosynthesis. In the following
50 years, with the progress of modern electronics and photooptics,
highly sensitive and fast fluorometers were developed which
contributed substantially to the elucidation of the basic mechanisms
involved in the complex process of photosynthesis. Chlorophyll
fluorescence always has been a pioneering tool. Many aspects which
eventually were analyzed in great detail by more specific methods,
were first discovered by chlorophyll fluorescence measurements.
Such discoveries are still taking place, presently mostly at the level
of regulation of the complex photosynthesis process under the
control of changing environmental factors. This still is a widely open
field of plant science, as only recently the instrumentation and
methodology for in situ fluorescence measurements and analysis has
become generally available. Progress in this field of research has
been greatly stimulated by the invention of the Pulse-AmplitudeModulation (PAM) measuring principle (see section 12.2 below).
The first PAM-101 Chlorophyll Fluorometer, with its accessory
modules 102 and 103, as well as the PAM-2000 Portable
Fluorometer have been successfully used all over the world, as can
be judged from the large number of publications based on
investigations carried out with these instruments.
The MINI-PAM differs from the previously issued PAM
fluorometers in that it is further miniaturized and optimized in order
to perform one particular type of measurement with the greatest ease,
accuracy and reliability, namely the determination of the effective
quantum yield of photosynthetic energy conversion, ΔF/Fm', the so57
CHAPTER 12
MEASUREMENTS WITH THE MINI-PAM
called Genty-parameter. In the following sections some background
information on this and other fluorescence parameters is given, and
special aspects on fluorescence measurements with the MINI-PAM
are outlined, in order to make optimal use of this instrument.
12.1 Chlorophyl fluorescence as an indicator of photosynthesis
Photosynthesis involves reactions at five different functional
levels:
• processes at the pigment level
• primary light reactions
• thylakoid electron transport reactions
• dark-enzymic stroma reactions
• slow regulatory feedback processes
In principle, chlorophyll fluorescence can function as an
indicator at all of these levels of the photosynthesis process.
Chlorophyll is the major antenna pigment, funneling the absorbed
light energy into the reactions centers, where photochemical
conversion of the excitation energy takes place.
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Fig. 9: Schematic view of primary energy conversion and primary
electron transport in photosynthesis. LHC, light harvesting
pigment-protein complex; P680 and P700, energy
converting special chlorophyll molecules in the reaction
centers of photosystem II (PSII) and photosystem I (PSI),
respectively; Pheo, pheophytin; DCMU, PSII inhibitor
(diuron); PQ, plastoquinone; PC, plastocyanin; Fd,
ferredoxin
The indicator function of chlorophyll fluorescence arises from
the fact that fluorescence emission is complementary to the
alternative pathways of de-excitation, which are photochemistry and
heat dissipation. Generally speaking, fluorescence yield is highest
when the yields of photochemistry and heat dissipation are lowest.
Hence, changes in fluorescence yield reflect changes in
photochemical efficiency and heat dissipation. In practice, the
variable part of chlorophyll fluorescence originates mainly in
photosystem II and excitation transfer to photosystem I may be
considered an additional competitive pathway of de-excitation.
Measuring chlorophyll fluorescence is rather simple: The
emission extends from 660 nm to 760 nm, and if shorter wavelength
excitation light is used, separation of fluorescence from the
measuring light is readily achieved with the help of optical filters.
The challenge arises with the wish to measure fluorescence in
ambient daylight and to use very strong light for the so-called
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'quenching analysis'. For this purpose the PAM measuring principle
has been developed which allows monitoring fluorescence against
106 times larger background signals (see 12.2).
From the viewpoint of fluorescence emission there are two
fundamentally different types of competing de-excitation processes:
• photochemical energy conversion at the PS II centers
• non-photochemical loss of excitation energy at the antenna and
reaction center levels
By both mechanisms, the maximal potential fluorescence yield is
'quenched' and, hence, 'photochemical' and 'non-photochemical
fluorescence quenching' can be distinguished. For interpretation of
fluorescence changes, it is essential to know the relative
contributions of these two different quenching mechanisms to the
overall effect. If, for example, fluorescence yield declines, this may
be caused by
• an increase of the photochemical rate at the cost of fluorescence
and heat-dissipation
• or an increase of heat-dissipation at the cost of fluorescence and
photochemistry
These two possibilities can be distinguished by the so-called
'saturation pulse method':
With a very strong pulse of white light the electron transport
chain between the two photosystems can be quickly fully reduced,
such that the acceptors of PSII become exhausted. Hence, during the
saturation pulse photochemical fluorescence quenching becomes
zero and any remaining quenching must be nonphotochemical. It is
assumed that changes in non-photochemical quenching are too slow
to become effective within the approx. 1 second duration of a
saturation pulse.
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Fig. 10:
On the basis of these considerations so-called 'quenching
coefficients' qP and qN were defined, which can be determined by
simple fluorescence measurements (see 12.3.6). For qP- and qNdetermination it is necessary to define the extremes of maximal and
minimal fluorescence yield, which are given in the dark-adapted state
(see 12.3.4). However, quenching analysis is not restricted to qP- and
qN-determination and very relevant information can be also obtained
without previous dark-adaptation of the samples. This is an important
point for field investigations, for which the MINI-PAM was
optimized.
In recent years, evidence from a number of research groups has
shown that the overall quantum yield of photochemical energy
conversion can be assessed by the simple expression:
YIELD = (Fm'-F)/Fm' = ΔF/Fm'
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This expression, which was introduced by Genty et al. (1989) is
identical to the YIELD-parameter measured by the MINI-PAM (see
12.3.7). With this fluorometer, YIELD-determination has become
exceedingly simple: The fiberoptics are held at short distance (ca. 10
mm) to a sample, and the START-key is pressed. Everything else is
proceeding automatically within seconds:
• the present fluorescence yield F is sampled
• a saturation pulse is applied
• Fm' is sampled (displayed as ... M)
• YIELD = (Fm'-F)/Fm' is calculated and shown on the LC-display
• the obtained data are stored in the MEMORY.
The simplicity of this measurement is contrasted by the profound
information it provides. In steady-state illumination, as prevailing
under field-conditions, the YIELD-parameter reflects the efficiency
of the overall process. Any change at the various functional levels
(outlined at the start of this section) will be reflected in this
parameter. The accuracy of this measurement is very high, and as
recordings are quick, very detailed information on the photosynthetic
performance of plants under varying environmental and
physiological conditions can be obtained.
For full assessment of fluorescence information, knowledge of
environmental parameters is required, in particular of light intensity
and temperature. For example, if the measured YIELD of leaf A is
lower than that of leaf B, this does not necessarily mean that leaf A is
photosynthetically less competent than leaf B. The difference could
as well arise from leaf A being exposed to stronger light or to a lower
temperature than leaf B. The MINI-PAM offers the possibility to
measure photosynthetically active radiation (PAR) and temperature
at the same spot of a leaf where also fluorescence is measured (see
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8.3), such that together with every YIELD-value also the
corresponding values of PAR and temperature are entered into the
file of automatically stored data. When PAR is known, the apparent
rate of electron transport (ETR) is calculated (displayed as ...E).
For assessment of overall photosynthetic performance,
measurements in the steady-state are most informative. On the other
hand, additional information on the various partial reactions can be
obtained from analysis of so-called 'induction kinetics'. Upon a darklight transition, fluorescence yield displays a series of characteristic
transients, the so-called 'Kautsky effect', which reflect the whole
complexity of the process. The rapid transients contain information
on primary electron transport reactions, while the slow transients
reflect reactions at the level of enzyme regulation. Analysis of the
slow transients is greatly facilitated by use of the saturation pulse
method, which allows to distinguish between the contributions of
photochemical and non-photochemical quenching.
Since the introduction of the PAM Fluorometer in 1985, there has
been a boom in chlorophyll fluorescence research, at the basic as
well as at the applied level. This is reflected in a large number of
publications, due to which there has been considerable progress in
understanding of the indicator function of chlorophyll, of
photosynthesis as such, and of the regulation of photosynthesis under
stress conditions. The review articles, and the original papers cited
therein, which are listed in the Appendix (section 13.5) cover a
representative part of the work which so far was carried out. This
literature may be useful to become informed in more detail about
chlorophyll fluorescence and possible applications of the PAM
Fluorometer.
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12.2 The PAM measuring principle
With conventional chlorophyll fluorometers, the same light is
used for driving photosynthesis and for exciting fluorescence.
Separation of fluorescence from stray excitation light then is
achieved by appropriate combinations of optical filters (e.g.
excitation by blue light and protection of the detector by a red filter,
which only passes the red fluorescence). Such conventional
fluorometers are of rather limited use for ecophysiological research,
as their function is severely disturbed by ambient daylight. In order
to distinguish between fluorescence and other types of light reaching
the photodetector, fluorescence excitation can be 'modulated': When
a special 'measuring beam' is rapidly switched on/off, the
fluorescence signal follows this on/off pattern and with the help of
suitable electronic devices the resulting modulated signal can be
separated. Standard devices for this purpose are lock-in amplifiers
which tolerate background signals several hundred times larger than
the fluorescence signal. For the extreme requirements of chlorophyll
fluorescence quenching analysis by the so-called saturation pulse
method (see 12.1), a new modulation principle was developed which
tolerates a ratio of 1:106 between fluorescence and background
signal. This measuring principle is patented (DE 35 18 527) and
licensed exclusively to the Heinz Walz GmbH.
The pulse-amplitude-modulation (PAM) principle displays the
following essential features (see also Fig. 11):
Fluorescence is excited by very brief but strong light pulses from
light-emitting diodes. With the MINI-PAM, these pulses are 3 μs
long and repeated at a frequency of 600 or 20000 Hz. The LED light
passes a short-pass filter (λ<670 nm) and the photodetector is
protected by a long-pass filter (λ>700 nm) and a heat reflecting filter.
A highly selective pulse amplification system ignores all signals
except the fluorescence excited during the 3 μs measuring pulses.
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The photodetector is a PIN-photodiode which displays linear
response with light intensity changing by factors of more than 109.
Hence, this measuring system tolerates extreme changes in light
intensity (up to several times the intensity of full sun light) even at
weak measuring light intensities. This property is essential for
correct determinations of photochemical quantum yield via the
fluorescence parameters Fv/Fm or ΔF/Fm' and of minimal and
maximal fluorescence yields, Fo and Fm (see 12.1 and 12.3.4).
Fig. 11: Schematic view of the PAM measuring principle
Due to the PAM measuring principle saturation pulse induced
fluorescence changes can be very selectively and reliably analyzed in
terms of photosynthetic activity. With the MINI-PAM, just like with
all PAM fluorometers, even small values of ΔF induced by a
saturation pulse can be relied on. This can be simply tested by
applying a saturation pulse (via START) to a fluorescing sample, like
the FLUORESCENCE STANDARD (Blue plastic filter) delivered
with the MINI-PAM, which is not capable of photochemical energy
conversion. With such a sample invariably YIELD = 0.000 is
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displayed. Such reliable performance, which is not possible with
conventional amplifier systems, is of particular importance when
photosynthesis yield is low due to stress conditions. In such cases it
is essential to be sure that total inhibition really is indicated by
YIELD = 0.000. These aspects are illustrated in Fig. 12.
Note: A small lowering of fluorescence yield observed upon
application of a saturation pulse to the FLUORESCENCE
STANDARD is a genuine effect which results from a transient
temperature increase within the sample (see 12.3.4).
Fig. 12: Comparison of fluorescence responses of photosynthetically
active (Nerium; Lichen, +water) and inactive (Lichen, dry)
plant samples with those of a fluorescing plastic filter. ML,
measuring light; SP, saturation pulse. The calculated values
of effective quantum yield of energy conversion in PSII
(YIELD) are depicted.
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12.3 Assessment of photosynthesis with the MINI-PAM: Outline
of the most important functions in practical applications.
As soon as the MINI-PAM is switched on, it continuously
monitors the fluorescence yield of a sample which is close to the
fiberoptics exit. In section 12.1 it was outlined, in which way
fluorescence yield relates to the effective quantum yield of
photochemical energy conversion. Assessment of this very
fundamental information is made automatically by two consecutive
measurements of fluorescence yield (initiated by START), one
briefly before and one during a short pulse of saturating light. The
effective quantum yield of photochemical energy conversion (Y,
YIELD) is then simply calculated from the equation Y = ΔF/Fm.
Although this sounds easy and straightforward, in practice certain
aspects must be taken into consideration to obtain optimal and
meaningful results (for a brief outline, see section 5). While it is
almost trivial that the actual measurement must be correct, it is also
important that the conditions are properly chosen to give meaningful
information. Both of these two aspects are dealt with in the following
sections, which outline the most important functions of the MINIPAM, corresponding to some selected points of the MODE-menu. A
short description of all 51 points of the MODE-menu is given in
section 7.
12.3.1 Maximal photochemical yield Fv/Fm
In green plants the maximal quantum yield of photosystem II is
observed after dark adaptation when all reaction centers are open (all
primary acceptors oxidized) and heat dissipation is minimal. Then a
saturation pulse induces maximal fluorescence yield, Fm, and
maximal variable fluorescence, Fv, such that also ΔF/Fm = Fv/Fm is
maximal. Fv/Fm, if properly assessed, is a reliable measure of the
potential quantum yield of PS II. It is lowered by all effects which
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cause inhibition of PS II reaction centers and increase of heat
dissipation. In this respect, photoinhibition is particularly relevant.
Phenomenologically, both an increase of Fo or a decrease of Fm may
contribute to a decrease of Fv/Fm = (Fm-Fo)/Fm. While an increase
of Fo points to photodamage, a decrease of Fm reflects enhanced
nonradiative energy loss (heat dissipation), which can be viewed as
an expression of photoprotection.
12.3.2 ML-BURST (menu point 5)
Plants can differ widely with respect to their requirements for
dark-adaptation. For some indoor potted plants less than 0.1 µmol
quanta m-2s-1 may already cause closure of PS II centers
accompanied by a fluorescence increase, whereas most outdoor
plants display close to minimal fluorescence yield and maximal
Fv/Fm in the steady-state at 10-40 µmol quanta m-2s-1. Even if a
sample is kept in absolute darkness, the actual fluorescence
measurement requires some excitation light. With the MINI-PAM,
under standard conditions this amounts to ca 0.15 µmol quanta m-2s1
. It can be decreased by lowering the measuring light intensity
(menu point 50: MEAS-INT) or by applying the 'burst mode' (menu
point 5: ML-BURST).
The burst mode is particularly useful for a quick check whether
the measuring light intensity is too high or not. By intermittent dark
periods the integrated intensity is cut to 1/5, which will result in a
lowering of fluorescence yield and an increase in Fv/Fm in very light
sensitive plants. In this case, the MEAS-INT could be further
lowered via menu point 50. Application of the burst mode has two
advantages: First, there is no loss in signal/noise ratio. Second, there
is no need to repeat AUTO-ZERO via menu point 2.
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12.3.3 AUTO-ZERO (menu point 2)
The MINI-PAM, like any other chlorophyll fluorometer, is not
absolutely selective for chlorophyll fluorescence but also shows a
small signal when no plant sample is in contact with the fiberoptics.
This false signal originates from traces of scattered measuring light
which reach the photodetector despite the blocking filters. Most
importantly, this signal is not dependent on properties of the
investigated sample and, therefore, is constant as long as the
measuring light intensity (menu point 50) and the gain (menu point
49) are not changed.
The false signal can be automatically subtracted from all
measured fluorescence signals by the AUTO-ZERO function (menu
point 2). For this purpose, the sample is removed and in menu
position 2 AUTO-ZERO is carried out via SET. Thereafter the signal
(F) without a sample fluctuates around 0 and sample specific
fluorescence can be assessed. Any changes in GAIN (menu point 49)
or MEAS-INT (menu point 50) lead to corresponding changes in the
offset voltage caused by the false signal. Therefore, in this case
AUTO-ZERO has to be repeated. If this is not done and a new
YIELD-determination is made via START, there is the warning?
NEW OFFSET?, which reminds the user to first carry out AUTOZERO (without sample) in order to determine YIELD correctly. If
the user prefers to keep the old offset value, the warning can be
overruled simply by pressing SET (while in menu position 1).
Any false signal which is not compensated by AUTO-ZERO
(menu point 2) or manually by ZERO-OFFS (menu point 44) will
lead to underestimation of Y (ΔF/Fm' or Fv/Fm). Normally, i.e. with
a leaf at 10-15 mm distance from the fiberoptics, the error is small
(approximately 2 %). The error can increase considerably, when
samples with low chlorophyll content and unfavorable geometries
are assessed. In such cases, the signals can be made large by
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applying maximal measuring light intensity and maximal gain.
However, in this way also the background signal is increased and
AUTO-ZERO becomes very essential. For example, in an
experiment with a 1 mm² piece of a leaf at maximal gain and
measuring light intensity quite reproducibly an Fv/Fm = 0.610 to
0.630 was measured, when no offset was applied. However, when
AUTO-ZERO was properly applied, Fv/Fm = 0.795 to 0.815.
12.3.4 Fo, Fm (menu point 25)
Fo and Fm are defined as the minimal and maximal fluorescence
yields of a dark adapted sample, respectively. Knowledge of Fo and
Fm is required for determination of the quenching coefficients qP,
qN and NPQ (see section 12.3.6). Fo and Fm determination is carried
out in menu position 25 via SET. Then in menu position 26 there is
automatic reset of qP to 1.000 and of qN to 000 and in menu position
27 NPQ is reset to 0.000. With all consequent applications of
saturation pulses (via START), calculation of the quenching
coefficients will be based on these Fo, Fm values, until they are redetermined via SET in menu position 25. As outlined in section
12.3.2, the threshold of light intensity below which a sample is darkadapted can vary considerably. In most plants Fm/Fo = 5 to 6, which
is equivalent to Y = Fv/Fm = 0.800 to 0.835. Such high values can be
measured only when true dark-adaptation is reached and the
measuring procedure is optimized as outlined in the preceding
sections 12.3.2 and 12.3.3.
For Fv/Fm, just as for YIELD-measurements in general, the
absolute signal amplitudes are of no concern, as long as Fo and Fm
are measured under the same conditions. However, measurements of
absolute signal amplitudes are important for full assessment of
photoinhibition (see 12.3.1) and also for calculation of the quenching
coefficients qP, qN and NPQ (see 12.3.6). It must be emphasized,
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however, that it is not a simple matter to compare absolute
fluorescence values of a sample measured at different times and
under different conditions. While it is almost trivial that the sample
must be in exactly the same position with respect to the fiberoptics
(e.g. in a suitable leaf-clip) and that the same settings of MEAS-INT
(menu point 50) and GAIN (menu point 49) must be used, it is less
obvious that the sensitivity of the fluorometer is affected by
temperature. A 1 °C increase results in an approximately 1 %
decrease in signal amplitude. This is due to the fact that the
efficiency of the light-emitting-diode, which provides the pulsemodulated measuring light, slightly drops with increasing
temperature. Hence, any internal heating of the fluorometer will lead
to a corresponding decrease of the signal amplitude (see section
12.3.5). To take this aspect into account, the MINI-PAM features
measurement of internal temperature (menu point 35), which is
automatically registered with every YIELD-measurement and stored
in MEMORY.
12.3.5 INT.TEMP (menu point 35)
As outlined in the preceding section, the output of the measuring
light LED is a function of temperature, a feature common to all
solid-state lamps. Intensity decreases by approximately 1 % per °C.
In practice, this has to be accounted for whenever signal amplitudes
are compared. It is of no concern for YIELD-measurements (signal
ratios ΔF/Fm' or Fv/Fm), except for a small local temperature
increase within the LED when pulse frequency is switched from 0.6
to 20 kHz during a saturating light pulse which affects selectively
Fm.
The internal temperature of the MINI-PAM, which is measured
in the optical compartment in the vicinity of the halogen lamp, is
displayed under menu point 35: INT.TEMP. It can increase
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considerably with prolonged operation of the halogen lamp,
particularly at the higher intensity settings and when a high ALFACT (menu point 16) is used. Therefore, actinic illumination time
(menu point 14: ACT-WIDTH) is limited to 5 min and not more than
2 min are recommended for recordings of LIGHT CURVES, which
involve 8 consecutive illumination periods (see section 12.3.9).
Nevertheless, internal temperature increases of 10-20 °C are not
uncommon, which will cause a decrease of measuring light intensity
in the order of 10-20 %.
Small temperature related decreases of measuring light intensity
also occur when the frequency is switched from 0.6 to 20 kHz. This
is normally the case during actinic illumination with the internal
halogen lamp. The extent of decrease depends on measuring light
setting (menu point 50: MEAS-INT) and the length of the period at
20 kHz. It can be ignored at all measuring light settings for times
below 2 s and amounts to approx. 1 % when a 3 s pulse is given at
ML-setting 12. Hence, the effect on YIELD-determinations can be
considered marginal. During prolonged 20 kHz operation at maximal
ML-setting, the effective intensity drops by 2-3 %. This effect is
reversible within a few minutes after returning to 0.6 kHz.
Changes in measuring light intensity induced by temperature
changes, just like any effect on the sensitivity and selectivity of
fluorescence measurements with the MINI-PAM, can be also
evaluated by monitoring the fluorescence signal of the
FLUORESCENCE STANDARD delivered together with the
instrument. This blue plastic filter (Roscolene Surprise Blue) emits
red fluorescence at an intensity similar to a leaf. As there is no
photochemical energy conversion, fluorescence yield of this sample
is constant during illumination, provided temperature is not
changing. Using the Leaf-Clip Holder 2030-B with the integrated
temperature-sensor, it can be readily shown that continuous actinic
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illumination and longer saturation pulses can induce temperature
increases within the fluorescence standard of up to 10 °C,
corresponding to a decrease of fluorescence yield of 4 %. Actually,
the heating during strong illumination also has a small effect on leaf
measurements, as also chlorophyll fluorescence is lowered by
approximately 0.4 % per °C. Therefore the intensity and duration of
saturation pulses should not be excessive. Otherwise there would be
some underestimation of YIELD which is, however, rather small. For
example, assumed the leaf surface heats up by 5 °C, instead of
Fv/Fm = 0.833 the measured value would be 0.830.
12.3.6 qP, qN and NPQ (menu points 26 and 27)
When a leaf is illuminated, its fluorescence yield can vary
between two extreme values, Fo and Fm, which can be assessed after
dark adaptation (see section 12.3.4). Any fluorescence lowering with
respect to Fm may be caused either by enhanced photochemical
energy conversion or by increased heat-dissipation (as compared to
dark state). As was outlined in section 12.1, saturation pulse
quenching analysis allows to distinguish between these two
fundamentally different types of fluorescence quenching. In brief,
photochemical quenching can be suppressed by a pulse of saturating
light (as photochemistry is saturated), whereas non-photochemical
quenching does not change during a saturation pulse (as changes in
heat-dissipation involve relatively slow processes). The quenching
coefficients are defined as follows (with Fm' being displayed as
...M):
qP =
Fm'− F
Fm'− Fo
qN =
Fm − Fm'
Fm − Fo
NPQ =
Fm − Fm'
Fm'
qP and qN can vary between 0 and 1, whereas NPQ can assume
values between 0 and approximately 10. The displayed quenching
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coefficients are meaningful only, if the values of Fo and Fm were
previously measured with the same sample at the same sensitivity,
i.e. with unchanged optical parameters, measuring light intensity (see
12.3.4) and gain.
The definitions of qP and qN imply that fluorescence quenching
affects only the so-called variable fluorescence, Fm-Fo, and not Fo.
In reality, at higher levels of qN (exceeding approx. 0.4) there can be
also significant quenching of Fo, resulting in the lowered yield Fo'.
This can be estimated upon light-off, when the acceptor side of PS II
is quickly reoxidised (within 1-2 s), whereas relaxation of nonphotochemical quenching requires at least 5-10 s. Far-red light,
which mainly excites PS I, can enhance QA-reoxidation and facilitate
assessment of Fo'. However, the MINI-PAM does not feature an
intrinsic far-red light source (as e.g. the PAM-2000). Therefore, it
should be realized that the measured values of qP and qN are valid in
first approximation only, in particular when strong energy-dependent
nonphotochemical quenching is given.
Fo-quenching is of no concern for NPQ-determination. The
definition of NPQ implies a matrix model of the antenna system
(Stern-Volmer quenching). With NPQ that part of non-photochemical
quenching is emphasized which reflects heat-dissipation of excitation
energy in the antenna system. NPQ has been shown to be a good
indicator for 'excess light energy'. On the other hand, NPQ is
relatively insensitive to that part of non-photochemical quenching
which is associated with qN-values between 0 and 0.4, reflecting
mainly thylakoid membrane energization. The different responses of
qN and NPQ are illustrated in Fig. 13 in which a plot of qN vs. NPQ
is shown. In this presentation, it is assumed that no Fo-quenching
takes place. When Fo-quenching affects qN-calculation, the
relationship extends to NPQ-values exceeding 4.
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Fig. 13: Relationship between qN and NPQ
12.3.7 YIELD-measurements of illuminated samples
With every application of START a YIELD-measurement is
carried out and on the LC-display of the MINI-PAM in the standard
menu position 1 the following signals measured in connection with a
particular saturation pulse are shown:
• F, fluorescence yield measured briefly before triggering of the
saturation pulse;
• M, fluorescence yield reached during the saturation pulse;
• Y, effective yield of photochemical energy conversion calculated
as YIELD = (M-F)/M;
• E, apparent electron transport rate (ETR) calculated as ETR =
YIELD * PAR * 0.5 * ETR-factor (displayed when Leaf-Clip
Holder 2030-B is connected)
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This and additional information is stored in the MEMORY (see
section 6) and can be transferred via the RS 232 interface to a PC
using the WinControl software (see separate manual) or the
PAMTRANS software (see 9).
Formally, there is no difference between YIELD-measurements
with dark-adapted or illuminated samples. In the former case, F and
M correspond to Fo and Fm (see 12.3.4) and Y corresponds to
Fv/Fm, the maximal photochemical quantum yield (see 12.3.1). In
practice, YIELD-determinations of illuminated samples are more
easy, as the effect of measuring light intensity can be neglected. On
the other hand, the interpretation of YIELD-data from illuminated
samples requires somewhat more background knowledge. Whereas
the dark-adapted state is well defined, there is an infinite number of
light states, mainly determined by quantum flux density (PAR),
illumination time, temperature and the physiological state of the
sample. Therefore, YIELD-measurements should be carried out at
defined light intensities and after defined periods of exposure to
these intensities.
In one of its most common applications, the MINI-PAM assesses
YIELD of plants in their natural light environment under steady-state
conditions. In this case, use of the Leaf-Clip Holder 2030-B with
automatic measurement of PAR and leaf temperature is very
convenient (see 8.3). For adjustment of defined PAR-values the
External Halogen Lamp 2050-HB (see 8.5) can be recommended.
12.3.8 ACT-LIGHT and ACT+YIELD (menu points 12 and 13)
For shorter periods of actinic illumination also the internal
halogen lamp can be used (see 8.1.3). As this leads to internal
heating, illumination times are limited to 5 min. However, even
much shorter times often are sufficient for reaching close to steady
state, when the sample before has been kept in ambient light for
some time. The actinic light can be turned on/off either by the double
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key operation ON + SET or in menu position 12 via SET. In the
latter case the remaining illumination time is displayed.
The MINI-PAM also features the possibility of combining actinic
illumination and YIELD-determination. In menu position 13: ACT +
YIELD, there is first actinic illumination and at the end of the chosen
period a saturation pulse is applied for YIELD-determination. This
function can be also started via the double key operation ON +
START.
The intensity of actinic illumination can be varied via menu point
15: ACT-INT which features 12 settings, with consecutive settings
differing by a factor of ca. 1.5. The effective quantum flux density at
the sample depends on the distance from the fiberoptics. Using the
Leaf-Clip Holder 2030-B at a standard distance of 12 mm the PAR
amounts to approximately 40 μmol quanta m-2s-1 at setting 1 and
2800 μmol quanta m-2s-1 at setting 12 (see list of relative light
intensities below). It should be noted that the absolute intensity is
also determined by battery voltage, and the quality of the individual
fiberoptics which may deteriorate with increasing time of use. Hence,
at a given setting of ACT-INT the effective intensity may vary and
for quantitative work parallel measurement of PAR is recommended.
For this purpose the Leaf-Clip Holder 2030-B in conjunction with
the LIGHT CALIB routine (MODE-menu point 8) is useful.
Intensities are approximately 20 % higher when using the 90°
fiberoptics adapter instead of the 60° adapter (see 8.3).
ACT-INT
Setting
1
2
3
4
5
6
7
8
9
10
11
12
Relative
Intensity
1.0
1.5
2.5
3.5
5.5
8.0
11.5
17
26
38
57
87
The range of PAR-values covered by ACT-INT settings 1-12 can
be shifted up or down via menu point 16: AL-FACT. In this way it is
possible to account for special measuring conditions (absolute actinic
77
CHAPTER 12
MEASUREMENTS WITH THE MINI-PAM
intensities at sample site being exceptionally low or high) or for
differences in light saturation properties of plants. The latter aspect is
particularly relevant in conjunction with the automatic recording of
light response curves (see 12.3.9). It is important to note that the
relationship between AL-FACT and PAR is non-linear. It depends on
the setting of ACT-INT (menu point 15) and also on battery voltage.
For example, at setting 10 and with a freshly charged battery the
PAR is increased by a factor of ca. 1.7 when AL-FACT is increased
from 1.0 to 1.5, and PAR is decreased by a factor of ca. 0.4 when
AL-FACT is decreased from 1.0 to 0.5. In practice, it is
recommended to measure the effective PAR with the Leaf-Clip
Holder 2030-B.
The duration of the actinic illumination periods is set via menu
point 14: ACT-WIDTH, with an upper limit of 5:00. For longer
illumination times an external actinic light source, like the External
Halogen Lamp 2050-HB is recommended. When several actinic
illumination periods are consecutively triggered, as with CLOCKoperation or LIGHT CURVE recordings (see 12.3.9), the ACTWIDTH should be small, in order to avoid excessive internal heating
of the MINI-PAM. In these applications it is limited to 3 min.
The ACT + YIELD function provides very essential information
on the state of the photosynthetic apparatus of a sample. At a given
photon flux density of photosynthetically active radiation (PAR),
which can be monitored by the micro-quantum-sensor incorporated
in the Leaf-Clip Holder 2030-B, the measured values of YIELD and
ETR of different samples can be directly compared and interpreted in
terms of relative electron transport rates. The efficiency of
photosynthetic electron transport can be limited by numerous steps in
the long sequence of reactions between the primary process of
photochemical energy conversion at the reaction centers and the
export of the assimilates out of the chloroplasts. In the steady state,
78
CHAPTER 12
MEASUREMENTS WITH THE MINI-PAM
the overall yield of assimilation is equivalent to the yield of energy
conversion at PS II. For a limitation to become apparent, the system
must be 'put under light pressure'. For example, if some stress factor
has caused a decrease in Calvin cycle activity, this will be only
expressed in YIELD or ETR, if a sufficiently high PAR is applied to
make dark enzymic steps of the Calvin cycle limiting. The maximal
YIELD of a dark-adapted sample, as measured by Fv/Fm (see 12.3.1)
and by YIELD-values at low PAR, will be affected only, if the stress
treatment has caused a limitation at the level of the primary reactions
of energy conversion (excitation energy capture efficiency and
charge separation efficiency at the reaction centers). This is, for
example, the case after photoinhibitory treatment. Photoinhibition
occurs, if a sample is exposed for longer time periods to excessive
light intensities. To what extent a given light intensity is excessive
depends on the physiological state of the sample and can be judged
by YIELD-measurements (see following section 12.3.9 on LIGHT
CURVES). A suppression of YIELD upon exposure of a sample to
excessive light does not necessarily reflect permanent damage, but
can also reflect a high potential for photoprotection by non-radiative
energy dissipation. The latter is associated with high values of qN
and NPQ (see 12.3.6).
12.3.9 LIGHT CURVE (menu point 17) and LIGHT-CURVE+REC
(menu point 18)
The recording of a LIGHT CURVE involves 9 consecutive
YIELD-measurements. The illumination series may start at
ACTINIC INTENSITY 1, 2, 3, 4 or 5 (set via the LC-INT function,
MODE-menu point 20). This feature allows to adjust the range of
applied intensities to the light adaptation properties of the sample
(sun or shade plant). When choosing the start-intensity, it should be
also considered that a lower intensity range reduces the danger of
79
CHAPTER 12
MEASUREMENTS WITH THE MINI-PAM
instrument overheating with longer illumination times. An alternative
possibility to vary the ACTINIC INTENSITY range is given by the
ACT-FACT function (menu point 16). This shifts all intensities up or
down.
Before starting a LIGHT CURVE recording, a sample should be
well adapted to a moderate light intensity, which is close to the light
intensity experienced by the plant in its natural environment. In this
way the requirement of long illumination periods for reaching
steady-state can be avoided. The length of the actinic-light-periods is
determined by LC-WIDTH (menu position 19). This is limited to
3:00 min in LIGHT CURVE recordings. With the 8 consecutive
illumination periods applied during a LIGHT CURVE it is advisable
not to exceed 2:00 min in order to avoid excessive internal heating of
the MINI-PAM.
A LIGHT CURVE is started either in menu position 17 via SET
or in any other menu position by double key operation ON + ∧. The
same commands apply for termination of a LIGHT CURVE. After
starting a LIGHT CURVE there is first a YIELD-determination in the
absence of actinic illumination for assessment of the maximal
quantum yield. The sample should be sufficiently shaded, such that
the external light does not contribute substantially to the PAR, which
is approximately 100 μmol quanta m-2s-1 at setting 3 of ACT-INT,
when the Leaf-Clip Holder is used under standard conditions.
Intensities are approximately 20 % higher when the 90° fiberoptics
adapter is used instead of the 60° adapter (see 8.3). Another
advantage of the 90° light incidence is a more homogeneous
illumination.
ACT-INT is automatically increased during the course of a
LIGHT CURVE and YIELD is automatically determined at the end
of each illumination period, the length of which is determined by the
ACT-WIDTH. This results in a total of 9 YIELD- and ETR-values,
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CHAPTER 12
MEASUREMENTS WITH THE MINI-PAM
which are stored in MEMORY (see 6) or transferred to a PC for
further processing. Light Curves are displayed under WinControl
(see separate manual).
Additional information on the dark-recovery of YIELD-lowering
during actinic illumination can be obtained by the function
L-CURVE+REC. This function can be started either via SET in
menu position 18 or by ON+∨. It can be terminated by the same
commands. The actual illumination program with L - CURVE + REC
is identical to that of a LIGHT CURVE. In addition, after termination
of the last illumination period, in the absence of actinic light the
recovery of YIELD in the dark is assessed by 6 consecutive
saturation pulses applied at 10 s, 30 s, 60 s, 2 min, 5 min, 10 min
after light-off. In this way, different types of non-photochemical
quenching can be distinguished which contribute to the lowering of
the PS II quantum yield. It is generally assumed that the rapid
recovery within the first 30-60 s reflects the disappearance of energy
dependent nonphotochemical quenching, in parallel with the
relaxation of the transthylakoidal ΔpH. The slower recovery within
the first 10-30 min is considered to reflect a change of energy
distribution in favor of PS II (so-called State Shift). The apparently
irreversible YIELD-lowering (with respect to the original dark state)
is expression of "photoinhibition".
LIGHT CURVES as measured with the MINI-PAM contain
somewhat different information than the conventional light response
curves. Correct measurement of the latter requires the attainment of
steady state at each PAR-value, which takes at least 10 min. LIGHT
CURVES recorded with short illumination times (down to 5 s;
so-called Rapid Light Curves, RLC) allow insight into the
physiological flexibility with which a plant sample can adapt its
photosynthetic apparatus to rapid changes of light intensity. Hence,
RLC contain information on induction as well as saturation
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CHAPTER 12
MEASUREMENTS WITH THE MINI-PAM
characteristics of photosynthesis. LIGHT CURVES measured during
the course of a day (e.g. triggered by the Repetition Clock, see
12.3.12) may show largely different characteristics due to the fact
that the physiological state of the photosynthetic apparatus is
regulated by environmental factors in a highly dynamic manner.
Whereas for proper recording of a conventional light response curve
it is essential that all conditions (like temperature, CO2concentration, humidity) are kept constant over extended periods of
time, LIGHT CURVES are sufficiently fast that they can characterize
a momentary state of a plant in a naturally changing environment.
12.3.10 YIELD- and ETR-averaging (menu point 11)
With normal samples under standard conditions the signal/noise
ratio obtained with the MINI-PAM is rather high, such that a single
measurement results in the reliable determination of YIELD and
ETR (see 12.3.7). In practice, the averaging function is most useful
in order to obtain representative information on the photosynthetic
performance of a heterogeneous sample. For example, repeated
measurements at one particular sample site may give YIELD- and
ETR-values fluctuating by no more than 0.001, whereas the values at
another site may differ by more than 0.1 units. This is particularly
true for outdoor measurements where the effective incident light
intensity depends strongly on sample position, possible shading etc.
In order to assess the effective quantum yield and the apparent
electron transport rate of a sample in a given situation under natural
conditions, the sample holder ideally must be attached such that there
is no sample shading. As this ideal can be only more or less
approached, unavoidably there is some variability in the results, and
averaging can be useful. The averaged data are not stored in the
memory. On the other hand, as every individual data set is stored in
MEMORY, which can be later transferred to a PC (see 9), users may
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MEASUREMENTS WITH THE MINI-PAM
prefer to evaluate statistical aspects of the data at a later stage,
applying standard programs. The optional WinControl software also
allows averaging of stored data in a most comfortable way.
Another case where averaging is advantageous relates to
measurements under extreme environmental conditions which cause
almost complete loss of variable fluorescence, i.e. when YIELD
approaches zero, while ETR may still be substantial due to high
PAR-values. Under such conditions, even the MINI-PAM becomes
limited by the signal/noise ratio in YIELD-determination, which can
be improved by averaging.
Before use of the averaging function, the SET-key must be
pressed in menu position 11 and AV. YIELD RESET must be
confirmed by pressing the ∧-key. Then with every application of a
saturation pulse the measured values of YIELD (Y) and ETR (E) will
be averaged until another reset is carried out.
12.3.11 INDUCTION CURVE (menu point 21) and
INDUCTION CURVE+RECOVERY (menu point 22)
Dark/light induction curves (Kautsky effect) contain complex
information on the photosynthetic performance of a plant at different
functional levels (see 12.1). By repetitive application of saturating
light pulses and quenching analysis additional information is
obtained which is essential for reliable interpretation of the Kautsky
effect. After a longer period of darkness, Calvin-cycle enzymes are
partially inactivated. They are light-activated during the first minutes
of illumination. During this induction period oxygen instead of CO2
serves as terminal electron acceptor. O2 dependent electron flow
(Mehler-Ascorbate-Peroxidase Cycle) as well as cyclic electron flow
at photosystem I create a large proton gradient, which will be used
for ATP-synthesis only after Calvin cycle has been light activated.
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MEASUREMENTS WITH THE MINI-PAM
This leads to strong "energy-dependent" nonphotochemical
fluorescence quenching during the first minutes of illumination
(characterized by low Fm'-values), which partially declines again
when CO2-fixation takes over and ATP is consumed.
In order to record an INDUCTION CURVE with the MINI-PAM,
a fixed geometry between sample and fiberoptics must be assured for
the duration of the recording. The recording is started by MODEmenu function 21: IND.CURVE. It is also possible to record the
light/dark recovery in addition to the dark/light induction
(22:IND.CURVE+REC). In this case information on postillumination reactions are obtained, in particular on the recovery of
various components of nonphotochemical quenching (see 12.3.9),
the extent of photoinhibition and also on dark electron flow between
stroma (or cytoplasma) and the electron carrier in the thylakoid
membrane.
Induction curves are either recorded via the analog output of the
MINI-PAM using a chart recorder or via the RS 232 interface using a
PC under WinControl-software. The latter offers the possibility of
online registration and display of various derived fluorescence
parameters, like effective quantum yield and quenching coefficients
(see separate WinControl manual).
Before recording of the actual induction curve, a single saturation
pulse is applied for assessment of Fo, Fm and Fv/Fm after dark
adaptation. This is a prerequisite for correct quenching analysis (see
12.3.1, 12.3.4 , 12.3.6). The delay between this saturation pulse and
onset of illumination can be varied (23: IND.DELAY); its default
value is 40 s. Another variable is the time interval between two
consecutive saturation pulses during actinic illumination (24: IND.WIDTH), with a default setting of 20 s.
Due to the outstanding role of molecular O2 during the induction
period, O2 partial pressures within the sample has a strong influence
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MEASUREMENTS WITH THE MINI-PAM
on all features of the the induction curves. This aspect is particularly
relevant for endosymbiotic phycobionts, as O2 is consumed by their
own and the host's dark-respiration and O2-diffusion is restricted (see
recent report by Schreiber, Gademann, Ralph and Larkum:
Assessment of photosynthetic performance of Prochloron in
Lissoclinum patella in hospite by chlorophyll fluorescence
measurements. Plant Cell Physiol. 38(8), 945-951, 1997).
12.3.12 Repetition Clock (menu point 28: REP-CLOCK and double
key function ON+MEM)
The Repetition Clock is primarily meant to trigger saturation
pulses for YIELD-determination at defined time intervals which are
set in menu position 30: CLK-TIME. The standard interval of 20 s is
appropriate for the recording of fluorescence induction curves with
repetitive YIELD-determination. The CLOCK can be started/stopped
in menu position 28 via SET. Then on the display the remaining time
to the next start of a function is shown. Start/Stop of the CLOCK is
also possible in other menu positions via the double key operation
ON+MEM.
Besides YIELD-measurements also other functions can be
repetitively triggered by the CLOCK. For this purpose the MODEmenu point 29 (CLOCK-ITEM) is provided which allows to choose
between:
1: SATURATION PULSE (SAT)
2: ACT+YIELD (A+Y)
3: LIGHT CURVE (LC)
4: L-CURVE+REC. (LC+)
5: INDUCTION-CURVE (IC)
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MEASUREMENTS WITH THE MINI-PAM
6: INDUCTION-C+REC. (IC+)
The CLOCK can be very useful for long term characterization of
the photosynthetic performance of a plant in its natural environment,
e.g. over the course of a day. As after every YIELD-determination
the corresponding data set is stored in MEMORY, in principle the
researcher just needs to start the CLOCK in the morning and collect
the data at night. In this context it is important to note that the full
capacity of a freshly charged battery allows approximately 12 hours
continuous operation of the MINI-PAM with standard YIELDdetermination every minute (total of ca. 720 saturation pulses). When
the CLOCK is running, the usual power saving function of AUTOOFF is disabled, with the consequence that there could be excessive
discharge of the battery. Therefore, in order to avoid battery damage
(see 11.1) the CLOCK is automatically switched off when battery
voltage drops below 8.5 V.
86
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APPENDIX
13 Appendix
13.1 Technical specifications
Photosynthesis Yield Analyzer MINI-PAM
Measuring light source: Light emitting diode, emission maximum at
650 nm; 12 intensity settings, standard
intensity 0.15 µmol m-2s-1 PAR;
modulation frequency 0.6 or 20 kHz; Auto
20 kHz function; burst-mode, 1/5
integrated intensity
Halogen lamp:
8 V/20 W blue enriched, filtered to give
λ<710 nm; 12 intensity settings, max. 6000
µmol m-2s-1 PAR with continuous actinic
illumination, max. 18000 µmol m-2s-1 PAR
during saturation pulses
Signal detection:
PIN-photodiode protected by long-pass
filter (λ>710 nm); selective window
amplifier (patented); sampling rate, 150
µs/point
Microcontroller:
CMOS 80C52
Memory:
Program memory, CMOS EPROM 32 kB;
Data buffer, CMOS RAM 128 kB,
providing memory for up to 4000 data sets
Measured and
calculated parameters: Fo, Fm, Fm', F, Fv/Fm (max. yield),
ΔF/Fm' (yield), qP, qN, NPQ, PAR and °C
(using Leaf-Clip Holder 2030-B), ETR (i.e.
PAR x ΔF/Fm')
87
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APPENDIX
Display:
2 x 24 character alphanumerical LCdisplay with backlight; character size
4.5 mm
User interface:
2 x 4 tactile keypad
Power supply:
Internal rechargeable battery 12 V/2 Ah,
providing power for ca. 1000 yield
measurements; automatic power/off when
not used for 4 min; Battery Charger MINIPAM/L
PC-terminal operation: Via RS 232 interface using special
command set; for remote control of all
functions
Data output and transfer: Analog output, 0-4 V; transfer on PC, via
RS 232 as ASCII-file using DOS or
Windows version of PAMTRANS Data
Transfer Program or WinControl Software
Dimensions:
19 cm x 13 cm x 9.5 cm (L x W x H)
Weight:
2.05 kg
Permissible ambient
temperature:
-5 to 45 °C
Windows-Software WinControl
for online PC-operation via RS 232-interface
Special functions:
88
Chart-display of fluorescence parameters
Induction curve registration
Light curve registration
Curve averaging
Display of saturation pulse kinetics
Report file
Data transfer
CHAPTER 13
APPENDIX
Fiberoptics MINI-PAM/F
Design:
Dimensions:
Weight:
Randomized 70 µm glass-fibers with steel
spiral envelope forming single plastic
shielded bundle with stainless steel adaptor
ends
Active diameter, 5.5 mm; outer diameter,
8 mm; length, 100 cm
0.18 kg
Battery Charger MINI-PAM/L
Power Supply:
Output:
Dimensions:
Weight:
100 to 240 V AC, 50/60 Hz
18 V/45 W
13.5 cm x 6 cm x 3.6 cm (L x W x H)
0.26 kg
Transport Case MINI-PAM/T
Design:
Dimensions:
Weight:
Plastic case with custom foam packing
42.5 cm x 34 cm x 13.5 cm (L x W x H)
1.9 kg
Leaf Clips:
Standard Clip
Design:
Positioning leaf surface at 60° with respect
to fiberoptics, for undisturbed incidence of
natural light
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APPENDIX
Dark Leaf Clip DLC-8 (optional)
Design:
Made of anodized aluminum with felt
contact areas and sliding shutter (closure);
weight, 3.6 g
Leaf-Clip Holder 2030-B (optional)
Design:
Micro quantum sensor:
Thermocouple:
Power supply:
Output:
Weight:
60° or 90° attachment of fiberoptics; for
simultaneous recording of PAR and °C at
measuring site
Selective measurement of
photosynthetically active radiation, PAR
(380-710 nm), 0 to 20000 µmol m-2s-1
Ni-CrNi, ∅ 0.1 mm, -20 to 60 °C
Via cable connecting to MINI-PAM
PAR and °C on LC-display of MINI-PAM;
push-button trigger-signal to start
saturation pulse for yield-measurement
0.35 kg
External Halogen Lamp 2050-HB (optional)
Wavelength:
<710 nm
Light intensity:
max. 3000 µmol quanta m-2s-1, stepless
setting
Power supply:
12 V/max. 1.6 A e. g. via Battery NP-3/12
Length of the
connecting cable:
120 cm
Weight:
0.25 kg
90
CHAPTER 13
APPENDIX
Micro Quantum/Temp.-Sensor 2060-M (optional)
for simultaneous recording of PAR and °C at measuring site
Micro quantum sensor:
Selective measurement of
photosynthetically active radiation, PAR
(380-710 nm), 0 to 20000 µmol m-2s-1
Thermocouple:
Ni-CrNi, ∅ 0.1 mm, -20 to 60 °C
Power supply:
Via cable connecting to MINI-PAM
Output:
PAR and °C on LC-display of MINI-PAM
Length of sensor cables: 30 cm
Weight:
0.22 kg
Miniature Fiberoptics MINI-PAM/F1 (optional)
for small spot measurements
Design:
Dimensions:
Single plastic-fiber with adaptor for MINIPAM
Active diameter, 2 mm; length, 150 cm
13.2 List of warnings and errors
Errors in MINI-PAM performance and warnings concerning suboptimal use of the instrument are signalled by messages in the upper
left corner of the display line. The following list briefly describes the
various error messages:
Err.
OVERFLOW: >3500
Maximal signal level was exceeded. The distance to the
sample should be increased. Alternatively, the GAIN (menu
point 49) or the MEAS-INT (menu point 50) may be
decreased. In the latter cases, the zero offset must be newly
determined (2: AUTO-ZERO).
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CHAPTER 13
Err.
APPENDIX
SIGNAL LOW: <130
Signal/noise ratio can be improved by increasing the signal:
For this purpose, decrease distance between fiberoptics and
sample; or increase GAIN (menu point 49) or MEAS-INT
(menu point 50). In the latter cases, the zero offset must be
newly determined (2: AUTO-ZERO).
Err.
LOW BATTERY
Battery voltage has dropped below 11.2 V which means that
only 20-30 further measurements are possible: Recharge
battery or connect external battery by special cable
(MINI-PAM/AK, optional).
Err.
? NEW OFFSET ?
Last measurement may be erroneous as GAIN (menu point
49) or MEAS-INT (menu point 50) was changed without
being followed by new zero offset determination (2: AUTOZERO). The warning can be overruled by pressing SET
while in menu position 1.
Err.
! CHECK BATTERY !
Battery voltage drops during application of a saturation pulse
below 8.5 V, which means that it is almost empty or too old:
Recharge or possibly replace battery.
Err.
MEMORY: 001
Maximal MEMORY-number of 4000 is reached. With further
measurements, the new data sets will replace the old data sets
starting from No. 1.
Additional warnings and information are given by messages in
the left corner of the upper display line:
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CHAPTER 13
APPENDIX
BAT
Battery voltage has dropped below 11.2 V: Be prepared that
the error message 3 (LOW BATTERY) will appear when
START is applied.
ACT
Actinic illumination is running.
A+Y
Actinic illumination with terminal YIELD-determination
(menu point 12) is running.
CLK
REPETITION-CLOCK (menu point 28) is running.
LC
Automatic recording of a LIGHT CURVE (menu point 17) is
running.
LC+
Automatic recording of a LIGHT CURVE + RECOVERY
(menu point 18) is running.
IC
Automatic recording of an INDUCTION CURVE (menu
point 21) is running.
IC+
Automatic recording of an INDUCTION CURVE +
RECOVERY (menu point 22) is running.
REC
Recovery part of LIGHT INDUCTION CURVE is running.
SAT
A saturating light pulse is applied for YIELD-determination.
13.3 PIN-assignments
"LEAF CLIP"
1
2
6
5
7
3
4
1:
2:
3:
4:
5:
6:
7:
+5 V
GND
⎫ Analog inputs Leaf-Clip Holder
⎬ 2030-B or Micro Quantum/Temp.⎭ Sensor 2060-M
Remote control button
-5 V
93
CHAPTER 13
APPENDIX
"RS 232"
1
5
2
4
3
1:
2:
3:
4:
5:
Not used
Not used
TxD
RxD
GND
"OUTPUT"
1: Signal output 0-4 V
2: GND
1
2
"CHARGE"
1
2
3
1: Charge input +18 V
2: GND
3: External input +12 V (max. 13.8 V).
ATTENTION: Internal battery cannot be
charged via this input.
13.4 List of commands for operation of MINI-PAM via
PC-terminal by user-written software
As described in chapter 10, the MINI-PAM can be operated by
remote control from a PC terminal. For this purpose a suitable
TERMINAL-program must be installed and the RS 232 interface
cable connected to the corresponding communication port. The
following commands are executed via 'Return'. Please note that only
low-case letters are effective.
94
CHAPTER 13
APPENDIX
In custom applications it should be made sure that at least 50 ms
elapse between two consecutively sent letters. The communication
has lower priority than the measuring routines and at higher rates
letters may get lost. For some commands the measuring program is
transiently stopped. Hence, data transfer should not occur during
measurements.
In addition, also the WinControl software is available, which has
been optimized for the communication between PC and MINI-PAM
and features a number of most comfortable functions for data
acquisition and analysis (see separate manual).
Command
?
a1/a0
aix
awx
a01/a00
af or afx
ay1/ay0
b
be1/be0
bp or bpx
c1/c0
ct or ctx
ci or cix
d or dx
Corresponding Description
point in
MODE-menu
37:
Date of Software version (current
EPROM)
12:
Act. light on/off
15:
Act. Intensity with setting x (x = 0
... 12)
14:
Act. Width with setting x (x = 10 s
... 5 min)
10:
AUTO-OFF on/off
16:
ACT.-FACTOR (x = 0.5 ... 1.5)
13:
Act. Light + YIELD on/off
BREAK, to stop all running
functions
Enable/Disable beep-function
Beep with length x (1 = 10 ms)
28:
CLOCK on/off
30:
CLOCK time (interval x = 10 ...
990 s)
29
CLOCK item (1..6)
48:
Damping setting (x = 1 ... 3)
95
CHAPTER 13
Command
dat or
dat(ddmmyy)
dl1/dl0
dsx
e
ea
ec1/ec0
ef or efx
APPENDIX
Corresponding Description
point in
MODE-menu
32:
Date (day month year)
9:
1:
11:
45:
f
1:
f*
1:
fmp
1:
fm
fo
fos
fz or fzx
fzs
g or gx
ic1/ic0
ic+1/ic+0
idx
iwx
l
25:
25:
25:
44:
2:
49:
21:
22:
23:
24:
1:
la1/la0
lg or lgx
lo or lox
lc1/lc0
lc+1/lc+0
le1/le0
lec1
lr
lw
6:
41:
40:
17:
18:
7:
8:
96
Display light on/off
Display MODE-menu point x
ETR (electron transport rate)
Averaged ETR (Leaf Clip)
Echo on/off
ETR-factor (defined as x)
Fluorescence yield before last
sat.pulse, F
Momentary fluorescence yield, F*
Max. fluor. yield during last
sat.pulse, Fm'
Max. dark adapted fluor., Fm
Min. dark adapted fluor., Fo
Fo-Fm determination
ZERO-OFFSET (defined as x)
AUTO-ZERO determination
Gain setting (x = 1 ... 12)
Induction Curve on/off
IC+Recovery on/off
Induction delay
Induction width
Light int. (PAR meas. with
ext.light sensor)
Light average on/off
Light gain (Leaf Clip)
Light offset (Leaf Clip)
Light curve on/off
Light curve + Recovery on/off
Ext. light sensor on/off
Light cal
Read light list
Write light list
CHAPTER 13
Command
APPENDIX
li or lix
lw or lwx
m1/m0
ma or max
mb1/0
mf1/0
mi or mix
me or mex
Corresponding
point in
MODE-menu
20:
19:
3:
51:
5:
4:
50:
38:
mez
39:
npq
o or ox
o+ or o+x
of
pao
pas
27:
paz
36:
qn
qp
s
si or six
sw or swx
t
ti
tox
tgx
tim(hhmm)
ub
us
26:
26:
ver
vx
47:
46:
1:
35:
42:
43:
31:
34:
34:
Description
Light curve start-intensity
Light curve step width
Measuring light on/off
Mark of sample (x = A ... Z)
ML-BURST function on/off
ML-frequency (20/0.6 kHz)
ML-intensity (x = 1 ... 12)
MEMORY-number (x = 1 ...
4000)
CLEAR-MEMORY (attention!
data will be erased)
NPQ-parameter
Display of data set Mem.x
Display of data sets from 1 to x
Display of data set format
MINI-PAM switched off
Display of present MINI-PAM
settings
MINI-PAM settings reset to
standard
Display of present qN
Display of present qp
Start saturation pulse
Sat. pulse intensity (x = 1 ... 12)
Sat. pulse width (x = 0.4 ... 3.0)
External temperature
Internal temperature
External temperature offset
External temperature gain
Time (hour minute)
Battery voltage
Battery voltage during last sat.
pulse
No. of program version
Voltage at channel x of A/D
97
CHAPTER 13
Command
y
ya
yn
yz
APPENDIX
Corresponding Description
point in
MODE-menu
converter (x = 0 ... 7)
1:
YIELD measured with last sat.
pulse
16:
Averaged YIELD
16:
No. of averaged YIELD-values
16:
Reset YIELD-averaging function
13.5 Selected reviews on chlorophyll fluorescence and related
topics
Allen JF (1992) Protein phosphorylation in regulation of
photosynthesis. Biochim Biophys Acta 1098:275-335
Allen JF (1995) Thylakoid protein phosphorylation, state 1-state 2
transitions, and photosystem stoichiometry adjustment: redox
control at multiple levels of gene expression. Physiol Plant
93:196-205
Baker NR and Horton P (1987) Chlorophyll fluorescence quenching
during photoinhibition. In: Kyle DJ, Osmond CB and Arntzen
CJ (eds) Photoinhibition, pp 145-168. Elsevier, Amsterdam
Björkman O (1987) Low-temperature chlorophyll fluorescence in
leaves and its relationship to photon yield of photosynthesis in
photoinhibition. In: Kyle DJ, Osmond CB and Arntzen CJ (eds)
Photoinhibition, pp 123-144. Elsevier, Amsterdam
Björkman O and Demmig-Adams B (1994) Regulation of
photosynthetic light energy capture, conversion, and dissipation
in leaves of higher plants. In: Schulze E-D and Caldwell MM
(eds) Ecophysiology of Photosynthesis. Ecological Studies 100,
pp 17-47. Springer, Berlin
98
CHAPTER 13
APPENDIX
Bolhar-Nordenkampf HR, Long SP, Baker NR, Öquist G, Schreiber
U and Lechner EG (1989) Chlorophyll fluorescence as a probe
of the photosynthetic competence of leaves in the field: a review
of current instrumentation. Functional Ecology 3:497-514
Bose S (1982) Chlorophyll fluorescence in green plants and energy
transfer pathways in photosynthesis. Photochem Photobiol
36:725-731
Briantais J-M, Vernotte C, Krause GH and Weis E (1986)
Chlorophyll a fluorescence of higher plants: chloroplasts and
leaves. In: Govindjee, Amesz J and Fork DC (eds) Light
Emission by Plants and Bacteria, pp 539-583. Academic Press,
Orlando
Butler WL (1978) Energy distribution in the photochemical
apparatus of photosynthesis. Annu Rev Plant Physiol 29:345378
Critchley C (1998) Photoinhibition. In: Raghavendra, A. S. (ed)
Photosynthesis: 264-272. Cambridge University Press
Dau H (1994a) Molecular mechanisms and quantitative models of
variable photosystem II fluorescence. Photochem Photobiol
60:1-23
Dau H (1994b) Short-term adaptation of plants to changing light
intensities and its relation to Photosystem II photochemistry and
fluorescence emission. J Photochem Photobiol B:Biol 26:3-27
Demmig-Adams B (1990) Carotenoids and photoprotection in plants:
A role for the xanthophyll zeaxanthin. Biochim Biophys Acta
1020:1-24
Demmig-Adams B and Adams WW, III (1992) Photoprotection and
other responses of plants to high light stress. Annu Rev Plant
Physiol Plant Mol Biol 43:599-626
99
CHAPTER 13
APPENDIX
Edwards GE and Baker NR (1993) Can CO2 assimilation in maize
leaves be predicted accurately from chlorophyll fluorescence
analysis? Photosynth Res 37:89-102
Falkowski PG and Kolber Z (1995) Variations in chlorophyll
fluorescence yields in phytoplankton in the world oceans. Aust J
Plant Physiol 22:341-355
Govindjee (1990) Photosystem II heterogeneity: the acceptor side.
Photosynth Res 25:151-160
Govindjee (1995) Sixty-three years since Kautsky: Chlorophyll a
fluorescence. Aust J Plant Physiol 22:131-160
Holzwarth AR (1991) Excited-state kinetics in chlorophyll systems
and its relationship to the functional organization of the
photosystems. In: Scheer H (ed) Chlorophylls, pp 1125-1151.
CRC Press, Boca Raton
Horton P and Bowyer JR (1990) Chlorophyll fluorescence transients.
In: Harwood JL and Bowyer JR (eds) Methods in Plant
Biochemistry, Vol 4, pp 259-296. Academic Press, New York
Horton P and Ruban AV (1992) Regulation of Photosystem II.
Photosynth Res 34:375-385
Joshi MK and Mohanty P (1995) Probing photosynthetic
performance by chlorophyll a fluorescence: Analysis and
interpretation of fluorescence parameters. J Sci Ind Res 54:155174
Karukstis KK (1991) Chlorophyll fluorescence as a physiological
probe of the photosynthetic apparatus. In: Scheer H (ed)
Chlorophylls, pp 769-795. CRC Press, Boca Raton
Kolber Z and Falkowski PG (1993) Use of active fluorescence to
estimate phytoplankton photosynthesis in situ. Limnol Oceanogr
38:1646-1665
100
CHAPTER 13
APPENDIX
Krall JP and Edwards GE (1992) Relationship between photosystem
II activity and CO2 fixation in leaves. Physiol Plant 86:180-187
Krause GH and Weis E (1984) Chlorophyll fluorescence as a tool in
plant physiology. II Interpretation of fluorescence signals.
Photosynth Res 5:139-157
Krause GH and Weis E (1991) Chlorophyll fluorescence and
photosynthesis: The basics. Annu Rev Plant Physiol Plant Mol
Biol 42:313-349
Lavorel J and Etienne AL (1977) In vivo chlorophyll fluorescence.
In: Barber J (ed) Primary Processes of Photosynthesis, pp 203268. Elsevier, Amsterdam
Lichtenthaler HK (1992) The Kautsky effect: 60 years of chlorophyll
fluorescence induction kinetics. Photosynthetica 27(1-2):45-55
Lichtenthaler HK and Rinderle U (1988) The role of chlorophyll
fluorescence in the detection of stress conditions in plants. In:
CRC Critical Reviews in Analytical Chemistry, pp S29-S85.
CRC Press, Boca Raton
Melis A (1991) Dynamics of photosynthetic membrane composition
and function. Biochim Biophys Acta 1058:87-106
Mohammed GH, Binder WD and Gillies SL (1995) Chlorophyll
fluorescence: a review of its practical forestry applications and
instrumentation. Scand J For Res 10:383-410
Moya I, Sebban P and Haehnel W (1986) Lifetime of excited states
and quantum yield of chlorophyll a fluorescence in vivo. In:
Govindjee, Amesz J and Fork DC (eds) Light Emission by
Plants and Bacteria, pp 161-190. Academic Press, Orlando
Papageorgiou G (1975) Chlorophyll fluorescence: An intrinsic probe
of phyotosynthesis. In: Govindjee (ed) Bioenergetics of
Photosynthesis, pp 319-371. Academic Press, New York
101
CHAPTER 13
APPENDIX
Pfündel E and Bilger W (1994) Regulation and possible function of
the violaxanthin cycle. Photosynth Res 42:89-109
Renger G (1992) Energy transfer and trapping in photosystem II. In:
Barber J (ed) The Photosystems: Structure, Function and
Molecular Biology, pp 45-99. Elsevier, Amsterdam
Renger G and Schreiber U (1986) Practical applications of
fluorometric methods to algae and higher plant research. In:
Govindjee, Amesz J and Fork DC (eds) Light Emission by
Plants and Bacteria, pp 587-619. Academic Press, Orlando
Schreiber U (1983) Chlorophyll fluorescence yield changes as a tool
in plant physiology I. The measuring system. Photosynth Res
4:361-371
Schreiber U, Bilger W and Neubauer C (1994) Chlorophyll
fluorescence as a nonintrusive indicator for rapid assessment of
in vivo photosynthesis. In: Schulze E-D and Caldwell MM (eds)
Ecophysiology of Photosynthesis, Vol 100, pp 49-70. Springer,
Berlin Heidelberg New York
Schreiber U, Hormann H, Neubauer C and Klughammer C (1995)
Assessment of photosystem II photochemical quantum yield by
chlorophyll fluorescence quenching analysis. Aust J Plant
Physiol 22:209-220
Schreiber U and Bilger W (1987) Rapid assessment of stress effects
on plant leaves by chlorophyll fluorescence measurements. In:
Tenhunen JD, Catarino FM, Lange OL and Oechel WC (eds)
Plant Response to Stress - Functional Analysis in Mediterranean
Ecosystems. NATO Advanced Science Institute Series, pp 2753. Springer, Berlin-Heidelberg-New York-Tokyo
102
CHAPTER 13
APPENDIX
Schreiber U and Bilger W (1993) Progress in chlorophyll
fluorescence research: major developments during the past years
in retrospect. Progress in Botany 54:151-173
Schreiber U and Neubauer C (1990) O2-dependent electron flow,
membrane energization and the mechanism of nonphotochemical quenching of chlorophyll fluorescence.
Photosynth Res 25:279-293
Schreiber U, Bilger W, Hormann H and Neubauer C (1998)
Chlorophyll fluorescence as a diagnostic tool: basics and some
aspects of practical relevance. In: Raghavendra A. S. (ed)
Photosynthesis: 320-336. Cambridge University Press
Seaton GGR and Walker DA (1992) Measuring photosynthesis by
measuring fluorescence. In: Barber J, Guerrero MG and
Medrano H (eds) Trends in Photosynthesis Research, pp 289304. Intercept, Andover, Hampshire
van Kooten O and Snel JFH (1990) The use of chlorophyll
fluorescence nomenclature in plant stress physiology.
Photosynth Res 25:147-150
Walker D (1992) Tansley Review No. 36. Excited leaves. New
Phytol 121:325-345
Wilhelm C and Büchel C (1993) In vivo analysis of slow chlorophyll
fluorescence induction kinetics in algae: progress, problems and
perspectives. Photochem Photobiol 58:137-148
Williams WP and Allen JF (1987) State 1/state 2 changes in higher
plants
and
algae.
Photosynth
Res
13:19-45
103
CHAPTER 14
RECHARGEABLE BATTERY
14 Rechargeable battery
The Photosynthesis Yield Analyzer MINI-PAM is equipped with
a rechargeable sealed-lead acid battery.
The life time is 1-3 years and it depends on the specific
application. A 10 °C rise of the temperature will decrease battery life
by approx. 25%. Near the end-of-life the standby capacity of the
battery will be reduced. When this reduction becomes persistently,
please replace the battery.
The battery cannot be overcharged, when the battery charger
supplied with the instrument is used! Do not use any other battery
charger!
Never store the instrument with a discharged or partially
discharged battery! It is recommended to charge the battery every
three months during the storage period.
ƒ
For optimum performance always recharge the battery
immediately after discharging!
ƒ
Never leave the battery in a discharged stage!
ƒ
Never short-circuit the battery terminals!
104
CHAPTER 15
WARRANTY CONDITIONS
15 Warranty conditions
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 one (1) year from the shipping date
(date on invoice).
The warranty is subject to the following conditions:
1. This warranty applies if the defects are called to the attention of
Heinz Walz GmbH, Germany, in writing within one year (1) of
the shipping date of the product.
2. 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.
3. This warranty shall not apply to any product supplied by the
Heinz Walz GmbH, Germany which has been subjected to
misuse, abuse, abnormal use, negligence, alteration or accident.
4. This warranty does not apply to damage caused from improper
packaging during shipment or any natural acts of God.
5. This warranty does not apply to underwater cables, batteries,
fiberoptic cables, lamps, gas filters, thermocouples, fuses or
calibrations.
To obtain warranty service, please follow the instructions below:
1. The Warranty Registration form must be completed and returned
to Heinz Walz GmbH, Germany.
2. 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, custom duties,
105
CHAPTER 15
WARRANTY CONDITIONS
and/or shipping costs incurred in returning equipment for
warranty service are at customer expense.
3. All products being returned for warranty service must be
carefully packed and sent freight prepaid.
4. 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.
106