Zeiss LSM800-2 Bedienungsanleitung

!!! IMPORTANT !!!

Please make sure that the chosen objective

corresponds to the one shown in the software.

If this is not the case, please restart the system.

(Otherwise the pinhole and pixel sizes, as well as the

sectioning thicknesses are all incorrect)

!!! IMPORTANT !!!

LSM800 upright has two new GaAsP PMT detectors.

As already explained during intros, the new PMT are very

sensitive and easy to oversaturate (and damage).

GaAsP detectors are shutting down if oversaturated,

terminating your experiment without further warnings!

Please use them correctly!

Zeiss LSM800-2 Upright confocal microscope August 6, 2019

I. System description:

Upright confocal microscope ZEN blue 2.6/ Hotfix-3

Motorized stage for multiple position imaging

Heating chamber

NO CO2 mixer

Multislide holder, dish holder, single slide holder

Objectives:

-Position1: Objective Plan-Apochromat 20x / NA 1.0 W DIC (WD=1.8mm)

-Position2: Objective Plan-Apochromat 10x / NA 0.45 Air (WD=2.1mm)

-Position3: Objective Plan-Apochromat 40x/1.4 Oil DIC (WD=0.13 mm)

-Position4: Objective Plan-Apochromat 20x / NA 0.8 Air

-Objective Plan-Apochromat 63x / NA 1.4 Oil (not mounted)

-Objective Plan-Apochromat 40x / NA 1.2 Water (not mounted)

-Laser lines: 405, 488, 561, 640 nm

Two very sensitive Gallium arsenide phosphide photomultiplier tube (GaAsP PMTs) detectors with free choice of the

spectral range. (Uses a tunable short pass and bandpass filter in front of the PMTs)

Be very careful with using it to prevent instrument damage! If oversaturated, detectors will switch off automatically.

An ESID detector for transmitted light.

Simultaneous detection of only two dyes possible, more than two have to be acquired sequentially

II. Starting up:

1. Switch on the multiple socket (1). Provides power to the computer and microscope controllers

2. Switch on SYSTEM (2).

3. Switch on COMPONENTS (3).

4. Make sure that lasers are on. Laser key should be in position “I”. (4)

5. Switch on the computer.

6. Turn on Cool LED (by pressing Big blue on/off button), if you need it for visual examination and don’t

forget to close the shutter when you don’t need it or at the end of your session.

7. Login: LSMuser (password: Useme!11)

8. Open ZEN Blue Software and press “ZEN System”

9. “Image Processing” is for offline analysis of your images, please do not use it here

III. Visual examination of the sample

1. Put mechanical switch on the right hand side of the microscope to the ”Eye” position.

2. Press “Locate” in the main microscope menu.

3. Since this is an upright system, changing objectives can only be manually done by rotating the objective

revolver. Rotation should work very easily when objectives are in the “up” position.

4. For imaging or visual inspection, the objectives must be lowered entirely with the black wheel in front of

the microscope stand.

5. Please make sure that the chosen objective corresponds to the one shown in the software. If this is

not the case, please restart the system. (Otherwise, your pinhole and pixel sizes, as well as the

sectioning thickness are all incorrect.)

6. If you wish to view your sample in Brightfield mode, press the “TL” button

7. For visual examination of fluorescence, press the buttons DAPI, GFP or RFP

IV. Acquiring confocal images

1. Set mechanical switch on the right hand side of the microscope to the “Camera” position.

2. Select “Acquisition” in the main menu of Zen. (Software will ask you about the mechanical switch,

Press”OK”.)

3. Go to “Smart Setup” add your dyes pressing “+” button (set the LUT) and configuration, you can choose

between simultaneous (fastest) and sequential (best signal) scan, press ”Apply” after choosing the

appropriate setup.

4. If you are using the detector for transmitted light: activate the ESID Detector represented in the light

path scheme. Make sure Köhler illumination is set properly if you are getting bad bright field images!

5. Go to “Live” acquisition modus and find the focus manually.

6. Set pinhole to Airy 1 in the channel dialog to achieve confocality.

7. Press the “Auto exposure” button and the software will adjust the gain of the PMTs but not the laser

power, according to the brightness of your dyes.

7.1. What Auto exposure does is to increase the PMT voltages until about 1% of all pixels are

overexposed. Gain cannot go below 500 V and above 900V.

8. In the channel dialog you can adjust the pinhole, gain and laser power manually to have the best image.

You can increase laser power by default only till 4,5%. If you need more power you need to enable

“Higher Intensity Laser Range”. Be aware that GaAsP PMT are very sensitive and if you will put to much

power, they shut down for the course of imaging (& do not turn on automatically). In that case you need

to stop acquisition and restart the imaging manually!

9. To zoom in your image, go to “Acquisition parameteres” at the drop down menu. With “Crop” you can

select the desired area of your image, then go to “Live” or “Snap” to apply, “Reset all” will remove the

zoom again. Depending on the scan speed, the lowest zoom is 0.5 and not 1.0. Before taking an image,

press “optimal” in the “acquisition mode” to adjust the optimal number of pixels and the pixel size

according to Nyquist sampling criteria.

Note: Acquiring more pixels than ”optimal” almost never makes sense, but feel free to decrease the

pixel number to increase acquisition speed at the cost of resolution.

10. To capture single images, click at ‘Snap’.

11. Setting up Z-stack:

-Enable Z-Stack and the Z-Stack dialog will appear

-Press “Live” and find first and last slices with the focus knob and select them in the software accordingly.

-The software will suggest the optimal slice number and interval which you can change manually

-Press “Start experiment”

12. Setting up time series:

-Enable “time series”

-Time series dialog will open

-Set cycle number and interval time

-Press “start experiment”

13. Tile Scan (in this mode you have: Tiles, Multipositions)

-Enable “tile scan”

-Tile scan dialog will open

-Set desired number of horizontal and vertical tiles

-You can choose different regions to scan. Choose regions and press button “+” to add it.

-Press “start experiment”

Advanced Set up allows you to do preview scan of region of interest.

14. Experiment designer.

-Enable “Experiment designer”.

-Experiment designer dialog will open

-Design your experiment using all options (z-stack, tiles etc). Choose how many blocks you want to have. You

can add loops.

-Press “Start experiment”

15. Data Saving:

Save your data to D: DATA folder in your own folder. Note that this computer is not backed Up. Please move

your data as soon as possible using portable storage devices or connect with intranet.

You can also use “Auto save”, your experiment and snaps will go directly to your folder.

V. Switching off:

1. DO NOT save any “Experiment” or overwrite the defult “Experiment” (marked with a home logo) as this setting

will be used in the next start up of the system and might cause potential damage to GaAsP PMTs.

2. Close the ZEN Software

3. Shut down the computer.

4. Turn off the cool LED (5)

5. Switch off COMPONENTS (3).

6. Switch off SYSTEM (2).

7. Switch off multiple sockets (1).

Laser Safety Instructions

During operation of class 3B and class 4 lasers red warning lights have to be switched on manually

Red warning light at the door of the room, containing laser-based equipment, prohibits the entrance

Optical path of the laser beam at all setups has to stay intact and should never be disassembled by a user. Users

are never permitted to disconnect optical connections (pipes, fibers etc), remove protective coverings or

disassemble any parts of the setups, especially those parts that are labeled with laser-warning signs.

User has to make sure, that objectives mounts are blocked by objectives or light- blocking plugs, before switching

the system on or starting the work

Any cleaning activities (objectives, stage cleanings) aswell as changing of objectives or filters have to be performed

only after blocking of the laser light is ensured. This can be ensured by closing the scanhead shutter or switching

off the laser.

Laser class-specific warnings at each setup have to be observed and considered

Eye contact with direct beam of Class 3B laser, or eye contact with mirror reflection from class 3B laser, should be

avoided at all times

Eye or skin contact with direct or diffuse light of Class 4 laser, should be avoided at all times

Laser safety goggles are situated at all workspaces and should be used in any situation where potential contact of

eyes with Laser light of the classes 3B or 4 is possible, according to the previous two points

Laser safety goggles have to be worn at all times of operation of Laser Class 4

Laser safety goggles have to be worn at all times of operation laser Class 3 at the optogenetic setups

Laser safety goggles are assigned to each setup and matched to the corresponding laser wavelengths. Matching

laser safety goggles should be used at all times, and should not be carried over between the setups

Only one person is allowed to be in the corresponding compartment during Laser Class 4 operation and

optogenetic setup operation

Users are not allowed to wear any reflective objects (rings, watches etc) during laser operation

Using of the equipment is only allowed after the introduction from a laser safety officer of IST Austria. Introduction

has to be done individually for each setup.

Changing experimental conditions, that involves changes in the laser application, have to be reported to the laser

safety officer prior to the start of experiment

Users have to understand that any violations against the instructed rules and also withholding information leading to

safety hazards will ultimately result in denial of admission to all laser equipped instruments at the IST Austria.

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