LEICA SP5 operating Manual
University of Zurich
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Center for Microscopy and Image Analysis
Leica SP5 Operating Manual
Table of contents
1. Starting procedure
1.1. Turing on the hardware…………………………………………………………2
1.2. Starting the software…………………………………………………………….2
2. Mounting and focusing of sample
2.1 The microscope…………………………………………………………………..3
2.2 Mounting of sample………………………………………………………………3
2.3 Selection of objective…………………………………………………………….3
2.4 Focusing of sample………………………………………………………………3
2.5 Illumination
2.5.1.Fluorescence……………………………………………………………….4
2.5.2. Bright field, Koehler illumination………………………………………... 4
3. Image acquisition in the confocal mode
3.1. Beam path settings……………………………………………………………5
3.2. Intensity adjustments……………………………………………………….…5
3.3. Set final scan parameters…………………………………………………….7
3.4. Execute experimet / save images……………………………………………8
3.5. Table image optimization……………………………………………………..8
4. Sequential scanning…………………………………………………………………….9
5. Acquiring a z-stack……………………………………………………………………. .9
6. Setting a timecourse…………………………………………………………………..10
7. Mark and Find / Tile Scan…………………………………………………………….10
8. APD……………………………………………………………………………………...10
9. Shutdown procedure…………………………………………………………………..10
10. Trouble shooting……………………………………………………………………….11
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University of Zurich
2
Center for Microscopy and Image Analysis
1. Starting procedure
1.1. Turning on the hardware
1. Turn on the fluorescence lamp
2. Switch on all three switches on the panel
3. Turn on the laser key
4. Login the computer
(username: f.lastname; note: f corresponds
to first letter of first name. Initial password:
dummy)
1.2. Starting the software
Klick twice into the icon “LAS AF”
The following windows appear
Select whether the system
should be operated in the
resonant or non-resonant mode.
Click on OK..
After the hardware is initialized, switch on the lasers in the software:
1. Klick into Configuration 2. Klick into Laser 3. Activate the lasers you require
.
Decide whether the table needs to
be initialized. Only necessary if you
need to define positions on the
stage.
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University of Zurich
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Center for Microscopy and Image Analysis
2. Mount and focus sample
The microscope:
2.1. Mounting of sample:
Put the slide up-side-down on the stage. If you are using immersion objectives, make
sure you use the appropriate immersion medium and never mix different media. Add
a drop to the objective or coverslip.
2.2. Selection of objective:
Select the objective directly on the scope by pressing the
objective changer buttons or more conveniently by the
software.
.
Note: Check objectives for the proper adjustment of correction collars and make sure
that the cap on the front of the objective is released.
2.3. Focusing your sample:
Start with the 10 x air objective to find the focus and to get an overview
of your sample. Focus your sample by moving the objective up with the
focus wheel or button on the microscope or with the z-drive on the
controller unit. Start in the coarse focus mode and fine tune with the fine
focus mode (toggle button on the controller unit).
Fix the focal plane:
To save your focal plane press button 1 and 2 (see illustration above) on
the right side of the stand together twice. The Z-position is now on 0.
Controller unit
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University of Zurich
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Center for Microscopy and Image Analysis
If you change than to another lens you just have to fine tune the focal plane.
Note: Always start with a low magnifying air objective. Be careful when working with
immersion lenses to not run into the sample.
2.4. Illumination:
Fluorescence:
Choose a filter by pressing the appropriate filter
button on the front of the stand.
Open the shutter.
Adjust the power of the fluorescence lamp by the
INT button on the left side of the stand
(see illustration below) or directly on the
fluorescence lamp.
Note: To avoid photobleaching work with an intensity as low as possible and close
the shutter when you stop observing your sample.
Bright field:
Choose bright field light by pressing the corresponding button
on the left side of the stand (BF, DIC, Phasecontrast).
Adjust the brightness with the INT button on the left side of the
stand or at the fluorescence lamp.
For DIC adjust the aperture diaphragm (AP) and Wollaston
Shear Control (wheel on objective turret) for optimal contrast.
For recording of bright field images adjust for
“Koehler illumination”:
1. focus specimen
2. fully close the illumination iris
3. focus condenser until diaphragm edge is in focus
4. move the illumination iris to the center with the 2 screws
5. open up illumination iris until it just fills the field of view
Note: It is always worthwhile to observe your cells in bright field mode to check
whether they are healthy.
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