Version:2-09
Confocal Protocol
Please save your data to Data disk (D)
1) Everything should be off (orange switch leftside scope,
mercury bulb, laser switches and red power switches).
2) Record mercury bulb hourly setting. Start mercury bulb
(Turn switch on).
3) Start microscope power switch (orange switch on left of
scope).
4) Turn on 3 red Power switches (far right), but not the
laser key(s).
5) Log in with lab's account name and password. Start up
Leica Confocal program( desktop) to record data,
Simulation (Start windowProgramssimulation) to
view old data.
6) Retract lenses using
lower button of
motorized focus control
(right side of scope).
Set view setting for
condenser light path (on
top of condenser) to
VIS for viewing with
eyepieces.
Set condenser to BF or
Phase1 as desired,
aperture wide open.
Using the OBJ window
in the ACQUIRE mode
in the software (lower
part of screen, line of processes), select the 10x
lens.
7) Gently push back light tower and mount sample on scope, coverslip side down. Pull
tower forward. Rotate light rheostat (lefthand scope, black wheel, behind on-of
switch) for comfortable brightness. There is no click on/off setting on this rheostat-watch the lamp values on the front L.E.D. Using the RH power focus buttons or the
cream focus knob, obtain a focussed image. The focus knob has 4 settings: S0 is finest
and S3 is coarsest (read value on front L.E.D). Change the sensitivity by pressing the
Step button on the microscope front L.E.D.
If you don't see anything, check that the confocal shutter (back, Rhand side
slider, by 'SCAN' wheel) is set all the way in.
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Version:2-09
8) For fluorescence, rotate brightfield power off, rotate cube
into position (lefthand side, recessed area below lenses--#3 is
for Green, #2 is for Red), pull out shutter slider knob,
(lefthand side, behind cube wheel). You should see
fluorescence. Be sure RH confocal shutter slider knob is set
to off (all the way in)
9) To move to a higher power objective, focus with the low
power lens, then go to the software and select the appropriate
objective. The lens turret will automatically rotate to the
correct power and move to approximate focus position. There
are two higher power oil immersion lenses (and one high
power water immersion lens). To add immersion fluid, press
the learn button (front panel)--the selected lens will go to the side, where you can add a
small amount of oil to the lens face. (Open the immersion oil bottle, wipe the paddle,
then allow the oil to accumulate at the bottom of the paddle. Gently touch the flat surface
of the paddle to the lens face and immediately press learn again. Your selected lens
should rotate back into position and approach focus.)
10) For Nomarski, non-confocal, push in the ICT/P slider on the left hand side of the
scope (analyzer in figure). Set the lens prism (on lens turret) to appropriate magnification
setting. Set the condenser filter to the appropriate magnification range, and confirm the
the polarizer filter on the top of the condenser assembly is in. For confocal Nomarski,
pull out the ICT/P slider, but leave other lens and condenser settings. For best results, be
sure you have set up the microscope for Koehler illumination! ( Focused, centered field
aperture, condenser aperture set for optimum contrast/resolution).
Once focused and ready to go, close fluorescence shutter slider, fluorescence filter wheel
to 4, pull out RH confocal shutter knob for scan on, condenser wheel to BF if Nomarski
is not required, set condenser light path knob to scan.
Turn on needed lasers (Green label 1, Red label 2, Far red 3. Right click Argon laser like
car to start). Appropriate yellow lights should be on. Turn up Level knob (by laser on
switches) to 9 am position.
LASERS ARE EXPENSIVE! HELP EXTEND THEIR LIFETIMES:
1) DO NOT TURN LASERS ON AND OFF. ONCE ON, LEAVE THEM ON FOR 3
HOURS. IF YOU LEAVE FOR A SHORT BREAK (LESS THAN 120 MINUTES),
TURN LEVEL TO MIN., THEN RESET TO 9AM WHEN YOU RETURN TO
WORK. FOR BREAKS OF LONGER THAN 3 HOURS, TURN OFF THE
LASERS.
2) DO DATA COLLECTION FIRST. WHEN COMPLETELY FINISHED
COLLECTING DATA, TURN OFF LASERS AND MANIPULATE YOUR DATA.
3) Check web calendar if anyone else will be using the lasers within 3 hours before
turning off lasers. Contact next users to confirm they will deal with lasers later.
TO MAKE CHANGES IN SETTINGS, YOU WILL NEED TO 'STOP' SCANNING.
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Version:2-09
Confocal software settings
Select ACQUIRE. Select BEAM. Select dyes of choice (doubleclick), and turn on
transmitted detector (ACTIVE check box) if desired.
Defaults: BIT to 8; MODE to XYZ for z series or single images; SPEED to 400 Hz;
SCAN to single arrow right
Set FORMAT for best speed vs resolution.
Set your PMT gains at the format you will scan at. Changing the format will
change the intensity. The third knob needs to be configured to Gain PMT Trans
You can use the AutoGain button to set approximate gain settings.
Select continuous mode and let it scan a time or two, then stop. Click on the
adjacent AutoGain button. The values will be automatically set. You can
then tweak if you aren’t happy.
Use the QLUT color table (left side of image window) to maximize signal (white)
with minimum green (underexposed) or blue (overexposed) IN YOUR AREAS
OF INTEREST.
Check through your focus range that the exposure is correct if you intend to
collect a z series. You should try to keep the gain values under 750.
Set PINHOLE to 1 Airy disc unit (if your gain is too high, using a larger Airy disc
setting will increase intensity (and decrease optical section thickness)
Set ZOOM to one
Focus sample. There are 2 focus controls: the extra fine z stage controller (z pos on
knob strip) and the stage control on microscope. Using the z knob on the strip, adjust
your focus. If sample position exceeds z stage range, set yellow line in the Z Series box
to the middle of the box and focus image with the scope focus knob. Once focused, you
should be able to use the z focus knob on the knob strip.
Use the PMT knobs on the strip module to increase or decrease the sensitivity. The
ZOOM knob will enlarge the image in the center of your field. The Z IN button allows
you to select a square portion of your field to selectively zoom in on. Zooming is the
equivalent to adding more pixels to your picture without increasing the file size. Adjacent
to the Z IN button in the laser selection window is a Z IN OFF button.
For improved images, set Line Ave to 4-8 and/or Frame Ave to 4-8. Line averaging can
photobleach sensitive samples, too many frame averages may lead to image blur. More
averages means longer time to collect images. The averaging only applies to Single Scan
or Series (not Continuous)
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