User Guide to the IBIF Leica TCS SP8 MP Confocal Microscope

User Guide to the IBIF Leica TCS SP8 MP Confocal Microscope
This version: 7.24.14.
Introduction
The IBIF confocal microscope is made available on a fee-for-use-hour basis to all users who have been
trained. Unlike our previous instrument, it has a number of hardware features, specifically the
objectives, the stage, and the condensers, that can be added or removed by the user, with the
permission of the Imaging Specialist. To minimize inconvenience to other users, and to reduce the risk
of inadvertent damage to the equipment, we ask that users obey two rules:
1) Leave the confocal as you found it. Replace and/or reset everything that you modified.
2) Be aware that other users may have failed to obey rule 1. Be alert, especially in regard to
mechanicals such as stage translation, condenser height, and objective rotation.
Thank you, and we wish you every success in your imaging. We are here to help.
The Confocal Microscope and Accessories
The instrument is a Leica TCS SP8 MP confocal
microscope, equipped for multi-photon confocal
microscopy. The components are the microscope
body, two automated stages, the scan head, the
electro-optical modulator (EOM), the monitor
and computer, and the Spectra-Physics Mai-Tai
tunable near-IR laser.
The items on the desk (along with the monitor,
keyboard, and mouse) consist of a confocal
controller, a microscope controller, a metalhalide power source (which performs the
function of the Hg bulb in the previous system),
and a controller for the Dodt contrast
enhancement system (which replaces the
Nomarski contrast enhancement method of the
previous system).
The unlabeled switch is a shutter for the
Mai Tai laser and should only be needed in
emergencies in which the beam is exposed.
1
Two photomultiplier tube (PMT) detectors and
two hybrid-PMT (HyD) detectors are available
for conventional (single-photon) confocal
microscopy.
Some cell-phone carriers use frequencies that
interfere with the HyD detectors. Switch your
phone off or leave it at the front of the room.
Two high-sensitivity Super HyD detectors are
installed for non-descanned (multi-photon)
confocal microscopy. These detectors are
electrically- and liquid-cooled for decreased
background. They should only be used in a
darkened room and with the microscope
covered by a dark room cloth (available).
The Super HyD detectors are also vulnerable to
cell-phone interference.
Behind the air table at the rear are three boxes of
instrumentation. The lower box is the Compact
Supply Unit, which houses the visible light lasers.
The smaller of the two boxes on top of the
Compact Supply Unit is the power supply and
cooling unit for the Super HyD non-descanned
detectors.
2
The microscope has no manual focusing
adjustment. Instead, it has two stages and two
computer-controlled focusing mechanisms. The
large X-Y stage performs lateral translation in all
modes. The nosepiece has its own focusing drive,
controlled from the microscope controller, or the
confocal controller, or from the software.
The smaller Super Z Galvo stage rides on
top of the large stage and includes the slide
holder. It is used for stacks. The large translation
stage can be used without the super X Galvo stage,
primarily for whole animal work or specialized
experimental chambers. Contact us for
instructions on how to do this.
3
System Specification
Lasers
The system has four visible-light lasers: 405 nm, 488 nm, 514 nm, 552 nm, and 633 nm. The 405 nm
should be used to excite DAPI or similar blue-emitting dyes, therefore use of the multi-photon laser will
not be necessary. The 488 nm and 552 nm lasers are for FITC and rhodamine bands respectively. The
514 nm laser can be used to excite YFP, while the 633 nm laser is for longer wavelength dyes such as
Cy5. All of the lasers are semiconductor types and can be switched on and off without special care.
All of the lasers are more powerful than those in the previous system (20 mW or
better versus 1 mW for the 543 and 633 lasers of the previous system). Users will find that only a few
percent excitation is required in most circumstances.
Optics
The available objectives (illustrated above, from left to right) are as follows:
Position
Objective
Magnification
n.a.
1
2
HC PL Fluotar
HC PL Apo
10x
20x
0.3
0.7
Working
distance
(mm)
11.0
0.59
25x
40x
40x
63x
0.95
1.30
1.10 Corr
1.40
2.5
0.24
0.65
0.14
3*
IRAPO
4
HC PL Apo
5*
IRAPO
6
HC PL Apo
*available on request
Medium
air
air,
coverglass
water
oil
water
oil
The air and oil objectives (1, 2, 4, and 6) are similar to those you have used in the past. The two water
objectives are especially good for deep-tissue imaging and are corrected for multi-photon excitation.
Note: the 40x and 63x objectives have spring-loaded front ends that can be inadvertently twisted and
locked in a withdrawn position. Handle them with care.
Disk Drives
The computer has two drives, a 120 Gb solid-state drive (C:) and a 1.8 Tb disk drive (D:). All data should
be saved to the D: drive.
4
Condenser
A transmitted light condenser is available if high-quality
visualization of unstained tissue or cells is required.
However, there is enough unfocussed light available in
transmitted light mode to enable simple localization of
specimens without a condenser. The Imaging Specialist
will demonstrate the user of the condenser if requested.
The condenser has a short working distance (1.0
mm) and very few options. Condenser centering is via
two screws, as in the Zeiss system. The condenser focus
knob is to the left side. The condenser cannot normally
be brought into focus, but as long as it is properly
centered it will function satisfactorily for the acquisition
of transmitted light images. The Dodt contrast system does not use the condenser.
If you have brought the condenser near to focus (for example, if trying to visualize unstained
cells), please remember to lower it before using the large X-Y stage, otherwise damage to the condenser
may result. For the same reason, install the condenser after the large X-Y stage has been initialized.
The condenser must be removed after use.
5
The Microscope Controller
Control of Stage Movement
Manual x, y and z stage control is via the knobs attached to the
microscope controller, or by the manual X-Y stage control on the
stage itself. The microscope controller moves the large X-Y stage
only. The knobs allow much finer control than the manual X-Y
stage control.
Touch Screen Control of Microscope Functions
The touch screens on the microscope controller are as follows:
Screen 1: Status and Illumination. Use to adjust
illumination intensity, and the aperture and field stops in
transmitted light (TL). These are in the microscope body,
not the optional condenser.
Screen 2: Illumination Method. Select TL or RL
(Reflected Light, i.e., epifluorescence). Open and close
the shutters to either path. In RL, select the filter cubes
(labeled GFP, RFP and DAPI) for green, red, or blue
emissions.
Screen 3: Objective selection. Some selections will pause
to allow time for oil or water application or removal. The
operation is completed by pressing the selection again.
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Screen 4: Nosepiece (i.e., objective) Z-control or stage X-Y
control.
Z-mode: The Home position is the highest point to which
the nosepiece can currently be raised. It is usually set at
the upper limit of travel of the nosepiece. The Focus Stop
position is the approximate focus position (actually, a
little above) for the 10x objective. Both positions are
indicated by – marks. The current position of the
nosepiece is indicated by the = mark and its distance
from the Focus Stop is indicated in mm on the display.
Press and hold the Home button to raise the nosepiece to the Home position. Press and
hold the up/down button to move the nosepiece to any position. Note that this button will allow
the nose piece to travel below the focus position. Careless use of it may drive the objectives into
the specimen.
The Home and Focus Stop positions should not normally be modified. See the section
below for a procedure to set or re-set them.
X/Y Mode: When X/Y mode is selected, the sensitivity of
the X-Y control knobs can be selected (coarse or fine).
Positions in the X-Y plane can be stored for future use.
Maintained touch in the screen will cause the stage to
translate to that position. If the condenser is installed,
be sure to lower it first.
Screen 5: Allows programming of any of the black
buttons for specific functions or combinations of
functions. See us before you program any of them.
Screen 6: Not normally needed by users.
7
Setting and Restoring Home and Focus Positions (Screen 5).
In some situations, for example when using the large X-Y stage in a lowered position, it may be
desirable to adjust the Home and Focus positions. Here’s how to do it.
First, select Screen 3 on the microscope controller display and select the 10x objective. Then
select Screen 4 and select Z-mode (top right).
Setting the Home position
Using the controls on the microscope controller, raise the
nosepiece all the way to the top, or to a level that allows
you to change your sample without interference from the
objectives. Note the Set/Clear Limits button (bottom left)
which has two parts, the Up/Down symbol
and the
word Home
.
On the Set/Clear Limits button, push the Home button
on the Set/Clear Limits button (it will turn red). The
button will now be labelled Set/Clear Home Position.
To set the Home position, hit the Set button (it will turn
red). The Home position indicator will appear.
To clear the Home position, hit the Clear button. It will
also turn red. The Home button will go gray.
8
Setting the Focus Stop position
Raise the nosepiece and set a glass slide specimen on the stage. If you do not have a suitable slide, there
are some H & E stained slides in the top drawer of the desk. The slides are autofluorescent to GFP or
YFP excitation, so you may use fluorescence as an alternative to transmitted light.
Manually lower the nosepiece until the specimen
is in focus through the eyepieces with the 10X lens. Raise
it a small amount.
On the Set/Clear Limits button, push the
Up/Down button (it will turn red). The button will now
be labelled Set/Clear Focus Position.
To set the current nosepiece position as the Focus
Stop position, hit the Set button (it will also turn red). To
clear the Focus Stop position, hit the Clear button. It will
also turn red. The Focus Stop position indicator will
appear.
If you have changed either setting, remember to
restore the Home and Focus positions for the next user.
9
Positioning a Slide
Raise the objectives using the microscope controller.
Displace the stage to the left manually or using the
stage X-Y manual control. For extra room, rotate the
objectives manually so that the 10x is over the stage. Be
sure that the clips are positioned at the edges of the
slide recess. If the clips are hard to move, use the hitech tool provided.
Place the slide in the slide recess. Usually, placing the
right side first is the most effective method.
Place the clips over the slide, using the tool if necessary.
Manually reposition the stage under the objectives.
Note: dishes can also be accommodated, subject to the
limitations of objective size and working distance. Ask us
for details.
10
Confocal Operation without Multi-photon
Users will not need multi-photon microscopy for DAPI excitation because of the availability of the 405
nm laser.
Metal-Halide Power Supply
Be sure that the metal halide power supply shutter
control button is set to remote (out).
Application Start
Turn on the computer, monitor, confocal power strip, and metal-halide power supply, in
any order. Double-click on the desktop LAS AF icon. Do not start LAS AF if the confocal
power strip is not on.
There are two questions to be answered during startup.
Optional selection of multi-photon laser. Select
MP_LASER_OFF.
Initialization of the large X-Y stage. Usually, select No.
Initialization of this stage is not necessary unless
you anticipate using the stage for tile scans. The manual
X-Y controls can still be used for specimen positioning.
If tile scans are to be used, protect the large X-Y
stage if necessary (raise the objectives, lower the
condenser if present), then select Yes. The large X-Y
stage will execute a series of X and Y movements before
returning to its home position.
11
LAS AF Application
The application screen will start up in the Acquire pane and will look like this:
Buttons and Sliders
Most functions are controlled by sliders. Click on the
slider button to change the slider value. Drag the button
or, for more precise adjustment, use the mouse wheel.
Some functions also allow keyboard data entry.
Confocal Controller
The confocal controller has six knobs that control
acquisition functions and two buttons that toggle the
current image/ detector combination. The knobs and
buttons can be operated while acquiring in Live mode.
Modifications to them will be reflected in the Acquire
pane. From left to right they are:
Left button – move the current detector/image one step to the left.
Right button - move the current detector/image one step to the right.
Smart Gain – adjust the gain of the current detector.
Smart Offset - adjust the offset of the current detector (if it is a PMT).
Scan Field Rotation - self-explanatory.
Pinhole Diameter (in Airy units)–use sparingly.
Zoom –a large range is available (0.7 to 40). An audible warning will sound when the usable
zoom has been exceeded.
Z position (in µm) – particularly useful when setting up stacks.
12
Program Panes
The program has four panes – Configuration, Acquire, Process, and Quantify, selected by the tabs at the
top left of the screen. Many Configuration pane options can be selected from the Acquisition pane,
which reduces the need to use the configuration window.
Configuration Pane
The Configuration Pane allows setting of multiple
hardware parameters. Most parameters can also be set or
modified from dialogs in the Acquisition Pane, so users
do not need to use its features often.
The Laser Configuration panel may be used to turn on or
off lasers. The lasers may also be set to Standby mode if
not required for a period.
The Hardware dialog is useful to select the acquisition
bit depth. The default is 8-bit, which is adequate for
image acquisition. If quantitative analyses are to be
performed, we recommend 12-bit acquisition. 16-bit
acquisition is available, but only for PMT channels.
The Configuration Pane also has a comprehensive dye
data base that is valuable when setting up novel
experiments.
The Objectives panel presents a window with useful
information about the objectives, such as numerical
aperture and working distance. It cannot be used to
change the working objective.
The Super-Z button allows selection of the working range
of the Super –Z Galvo stage. We recommend leaving it at
0.5 mm.
13
Acquisition Pane
Selection of Configuration
Sixteen standard configurations are available to all users
from the pull-down menu above. Please don’t modify
them! You can also create your own configurations and
store them.
Acquisition Mode
The acquisition mode defaults to xyz, with z = 1, that is, a single
frame. Other modes are discussed later in this document.
14
Acquisition Rate
The acquisition rate window selects the acquisition scan
parameters, including the number of pixels in the image
and the scan speed, via pull-down menus. We
recommend 400 Hz as an initial rate.
The image scan may be zoomed down to 0.7 and up to
20.
Averaging may be by line or by frame, using pull-down
menus – we recommend line averaging and no more
than 4 averages.
The scan orientation may be controlled using the
rotation slider. The image area may also be translated in
the directions indicated by the rosette arrows. Large
lateral translations at low zoom will move the scan range
off axis and are therefore not recommended. Move the
stage instead.
Pinhole
There is only one pinhole. Its diameter is determined by
the longest emission band and defaults to 1 Airy disk. It
may be modified by clicking and dragging on the pinhole
button.
Lasers
The visible light lasers must be enabled by clicking on
the ON/OFF button. The lasers may be selected using
the laser menu in the Configuration Plane, or by clicking
on the ‘+’ sign. Click and drag on the button to adjust
power level.
15
Objectives
Objectives may be selected by the pull-down menu. The
objectives will be raised and lowered as part of the
rotation. The multi-function plate (MFP) should be set to
substrate for most applications. Use Automatic
determination of the beamsplitter.
Selecting the Fluorescence Detectors
Up to four detectors may be selected simultaneously. The
detector type (HyD or PMT) is indicated in the top left of
each detector window. The detector is selected by
clicking on the on/off button. The color of its display
output is selected from a pull-down menu next to the
type indicator. HyD detectors have a gain selected by
clicking on its slider button. PMT detectors have both
gain and offset functions, controlled by clickable sliders.
Leica recommends a starting offset of -0.3%.
Modifiying the Detector Bandwidth
The detector bandwidth may be modified in either
direction by simply clicking on and dragging its edges.
The minimum bandwith is 5 nm. Adjacent detector
bands may not overlap.
Selecting the Transmitted Light Detector
The TL detector is selected by clicking on its ON/OFF box. It is
a PMT and is responsive up to 900 nm. Like the other PMTs it
has gain and offset controls. Proper centering of the condenser
aperture is required for optimum effectiveness. The condenser
need not be focused but it should be close to the specimen.
Use the Dodt controller to modulate the contrast. The
optimum Dodt setting when viewed with the eyepieces is not
necessarily the same as the optimum setting when using
confocal.
Acquisition Start Buttons
At the base of the pane are four action buttons. At left, the
Autofocus button should not be used. The Live button initiates
scanning at the indicated rate and format until it is pressed
again.
16
At right, the Capture Image button is used to acquire a single
image. The Start button is used to initiate acquisition of a stack
or a sequence.
Display
The displayed image is rotated 90° anti-clockwise with respect to the image seen in the objectives. Thus
x-axis stage translation moves the image vertically on the screen, and y-axis stage translation moves the
image horizontally.
Displaying Results
The look-up table LUT used to display the results may be
changed using the button at the top left of the display window.
The first click selects a range-finding LUT for the active window
that can be used to set gains and offsets. The second click
changes the display in all active windows. A third click returns
the LUT to the normal type.
The range-finding LUT is different to the one used by LSM. The LSM range-finding LUT used
blue for below range, red for above range, and white for within range.
below range
within range
above range
LAS AF uses green for below range, blue for above range, and red for within range.
below range
within range
above range
17
Programming a Stack
Programming a stack works similarly to the Mark
First/Last mechanism used in Zeiss LSM. It requires the
following steps:
1. With Live acquisition going, adjust the focus to
the top of the stack. Use the Confocal Controller,
or the microscope controller fine focusing knob.
Fine adjustments may be made using the
clickable wheel.
2. Click on Set Start.
3. Re-adjust the focus to the bottom of the stack.
4. Click on Set End.
The number of steps and the step size will be
automatically computed based on the longest
wavelength emission band. It can be modified by clicking
on Nr. of Steps or z-step size.
The diagram indicates the size and spacing of the
stack and the current stage position. It may be re-scaled
for better viewing using the slider provided.
Clicking on the double-vertical arrow button at the
top moves the stage to the middle of the stack.
Clicking on the garbage can icon will delete the
stack. This should be performed before creating a new
stack.
Use of the z-Galvo stage or the nosepiece for vertical
movement is controlled by the pull-down menu. The zGalvo stage is more precise.
Leave Galvo Flow off.
Acquiring from a Region of Interest
Unlike in Zeiss LSM, images can be acquired from a region of interest (ROI) alone, or from an ROI with
different conditions for an ROI and the background.
18
Click on the ROI button to set the foreground conditions. Acquisition conditions within the ROI
will be as specified in the Detectors panel.
Draw the ROI using the tools above the image window. In this example, a square ROI has been
selected.
To set the background conditions, click on the Background button.
19
Alternate Acquisition Modes – Sequential
Click on the right-most icon in the Acquisition Mode menu.
The Sequential Mode menu appears at the end of the acquisition
parameters window. Click ‘+’ to add sequential events, ‘-’ to remove
them. Up to six sequential acquisition events are possible. Events may
be any of the available modes, e.g., xy, xyz, xyt, xyλ. Events may be
acquired line-by-line (indicated by arrow). This is the preferred
method, because it involves less switching of the scan components.
However, if the detector bandwidth or the average or accumulation
number changes between events, use “Between Frames”. If one or more event is a stack, use “Between
Stacks”.
20
Alternate Acquisition Modes - Line Scan
21
Alternate Acquisition Modes - Spectral
Select xyλ or similar acquisition mode.
The laser and detector section of the acquisition pane
will appear as shown here.
The number of bands and the bandwidth is set in the lambdaScan Range Properties window. The maximum spectral range is
380 nm to 785 nm. The shortest-wavelength laser is 405 nm, so
the shortest practical wavelength is 420 nm, unless multiphoton excitation is used. The minimum bandwidth is 5 nm.
The minimum step between bands can be less than 5 nm. At 5
nm spacing, the maximum number of bands is 81. Any one of
the four descanned or the two non-descanned detectors may be
selected as the detector.
22
Alternate Acquisition Modes - Time Series
Select xyt or similar acquisition mode.
23
Alternate Acquisition Modes - Tile Scan
Select Tile Scan in the Acquisition Mode menu. The Tile Scan
window will appear below in the Acquisition window.
Note: Tile Scan should not be combined with image rotation or
erroneous results will be obtained.
Tile Scan Window
The Tile Scan window features an X-Y map showing the current
position of the stage. The scale is in mm, and the X-axis is
vertical, as for the image display. The map scale can be
expanded or contracted using the button and slider below the
map.
Above the map are four buttons. The left-most button
selects the current position for storage. Multiple positions can
be stored. The current position number is reflected below the
map. The second button deletes the current stored position, as
reflected by the position number below the map. The third
button deletes all currently-stored positions. Clicking on the
right-most button opens a stage configuration menu
The simplest method is to select Auto Stitching, with
Smooth off.
Stage Configuration Menu
Note that in the default condition, the X- and Y- axes are
inverted in sign (corresponding to the inverted
microscope image), and that the X and Y axes are
exchanged.
When Auto Stitching is selected, the percent of
overlap is selected in this menu. We find that 10%
overlap works satisfactorily without requiring excessive
sampling or computation time.
24
Setting up a Tile Scan
The approach to setting up a tile scan is different from that in Zeiss LSM.
Step 1: Click on the Select Position button to select the current position as Position 1.
Step 2: Move the stage to a position at one extreme of the area to be tiled. Use the microscope
controller X-Y controls to move to the new position, while observing through the eyepieces or
observing the display in Live mode. Click on the Select Position button to select the current position
as Position 2. The map will display a map indicating the number of tiles to be scanned. In this
example, a 2 x 1 tile map is to be acquired.
Stage 3: To expand the tiled area, move the stage to a new position at an orthogonal extreme, as
before. Click on the Select Position button to select the current position as Position 3. The map will
display a revised map indicating the expanded number of tiles to be scanned. In this example, a 3 x
3 tile map is now to be acquired.
Click on ‘Start’ to begin acquisition of the tiled scan. If ‘Auto Stitching’ is selected, after
acquisition is completed, the images will be automatically stitched together to form a composite image.
Combining Tiling with Stacks
Stacks may be combined with tiles and with averaging. Be sure to check that the stack dimensions are
appropriate at all locations of the tile scan.
25
Alternate Acquisition Modes – Mark and Find
Mark and Find enables you to store multiple locations on your
specimen so that you can return to them individually. It
functions much as the way you store locations in the X-Y plane
using Screen 4 of the Microscope Controller. However, it has the
advantage that the stored locations can be employed in a
sequential acquisition.
Setting Up a mark and Find List
The window shows an X-Y map, as in Tile Scan. Remember that
the X-axis is vertical, as in the Display. Move the stage using the
Microscope Controller X-Y controls, observing either via the
eyepieces or in Live mode.
At each position, find the appropriate focus. Click on
Select Position to store the position. The Position Number
window will increment. You may delete the current position
using Delete Current Position, or delete all currently-defined
positions using the trash can. You may also re-define the current
position.
A family of positions can be named and saved using the
floppy-disc icon (second from left). The left-most icon enables
you to recall a saved list of positions.
Use the pull-down list of position numbers to select a
new position. The stage will move to that position and the focus
position will be adjusted accordingly
Combining Tiling with Sequences
When defining a sequence involving changing X-Y position,
remember to select the appropriate position number when
defining the sequence event.
Combining Tiling with Stacks
A stack acquisition may be defined in a sequence acquisition. The stack may be different for each
position in a sequence. If the stack is to be the same, check the Same Stack for All box at the bottom of
this window.
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Storing Experiments
Click on the Experiments tab to show a list of completed
experiments.
Double-click on an experiment to inspect the result or analyze
it. Right-click on an experiment or sub-experiment to change its
name.
Use the Save button below in the Experiments window to save
experiments. You will also be prompted to save unsaved
experiments on shutdown.
27
Using the Experiment Metadata
If you desire to repeat the acquisition parameters of a
previous sub-experiment, you can do so by two different
methods.
If you are confident that the sub-experiment represents
the correct parameters
Load the experiment if it is not already loaded. Click on
the sub-experiment.
Select the Apply button at the base of the Experiments
window to apply the parameters.
If you wish to check the parameters first
Select the file, and right-click on it. A panel will appear
displaying the acquisition parameters. You may scroll
down the list of parameters to inspect them.
Click on the “Apply Settings” button at the bottom left of
the window to apply them.
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Note - Not all parameters will be applied.
These parameters are applied from an image file:




Laser status, including multiphoton (assuming multiphoton laser is on)
Multiphoton gain and offset
All detector settings (including acquisition type for HyDs - Standard vs Photon-Counting)
Pinhole diameter
These parameters are NOT applied:









Bit depth (important)
Format (pixel-by-pixel size)
Acquisition speed
Zoom
Number and type of average
Stage position
Z-axis position
Stack parameters
Tiling parameters
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Process Pane
30
Quantify Plane
31
Confocal Operation with Multi-photon
Detector Filters and Barrier Filter
The Super HyD detectors have a 620 nm barrier filter in front of them to protect against reflected IR
light. For bandwidth selection, you will need a suitable filter in front of the detector or detectors. Ask us
how to set this up.
Start Up
It is not necessary to power down the computer or the microscope when switching from
MP_LASER_OFF to MP_LASER_ON operation. However, certain steps must be followed for the
correct sequence.
Switch on the Electro-Optical Modulator.
The modulator is ready for use immediately, but does not
achieve full modulation depth for about 15 minutes. Switch it on
first.
Switch off the Compact Supply Unit at the back of the table.
Switch on the Super-HyD detectors power and cooling unit.
Switch the Compact Supply Unit back on.
Application Start - Select MP_LASER_ON.
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Pinhole
The pinhole is not in the emission path for the non-descanned detectors, so it can be ignored. Operation
of the confocal with multi-photon excitation and descanned detection is ineffective and should be
avoided.
Enabling the MP Laser
Unlike the semiconductor lasers, the MP laser must be
turned on in the Configuration pane.
Setting up the MP Laser
In the Acquire pane, first click MP on. Then click on the ‘+’ button to
open the MP laser window.
The MP Laser Window
Select the laser wavelength. The laser will take 2-3
minutes to go from ‘CW”, with little or no power, to
‘pulsing’, with full power (2-3 W). T0 open the shutter,
press and hold the shutter button.
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Dispersion Compensation
Dispersion compensation of the near-IR laser is set in the GDD
pull-down panel. Select the appropriate objective form the table.
The default (zero GDD) is adequate for multi-photon operation.
The fine tuning slider can be adjusted for special circumstances
(e.g., operation in deep tissue).
Setting the NDD Detectors
Starting up in MP_LASER_ON mode will enable the NDD
Super HyD detectors. These are controlled using a single gain
slider. The NDD detectors are very sensitive. Use low gain and
low laser power initially. Operate only in a darkened room. A
darkroom cloth is available to mask stray light.
Managing MP Laser Power
The EOM window, which controls MP laser power, has
four slider controls.
On the right, the EOM slider must be set to 1 to permit
near-IR excitation to reach the specimen. The offset
parameter is set by the application as a function of
wavelength and should not be modified.
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On the left of the window are two controls that are
misleadingly labeled. They are, effectively, coarse (left)
and fine (right) control of laser intensity at the
specimen. The number at the base of the left slider is the
excitation wavelength, in nm.
Best practice is initially to set the coarse slider to zero
and the fine slider to a low value. Using the Live mode,
increment the coarse slider slowly until an image is
obtained. Adjust the fine slider to obtain the optimum
image.
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Shut Down
The order is not important.
Switch off the EOM controller and the Super HyD power supply, if used.
Close the application – you will be prompted to save the files that you want to save. Save to
folders on drive D:
Log off the computer.
Switch off the confocal at the power strip.
Don’t forget to restore the Home and Focus levels if you have changed them. Replace the
condenser and any non-standard objectives in the drawer.
Ejecting Storage Devices
When ejecting your stage device, be careful not
eject the F: drive. This drive is the license dongle
that permits operation of the LAS AF software.
Ejecting it will prevent other users from using
LAS AF.
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