Zeiss LSM800-02 Bedienerhandbuch

Zeiss LSM800-02 + Airyscan confocal microscope 20/11/2019

System description:

I. Upright confocal microscope ZEN blue 2.6 HotFix-8

II. 2 GaAsP +1 AiryScan detector (GaAsP)

III. Motorized stage for multiple position imaging

IV. Heating chamber

V. NO CO2 mixer

VI. Multi-slide holder, dish holder, single slide holder

Objectives:

-Position1: Objective Plan-Apochromat 10x / NA 0.45 Air (WD=2.1mm)

-Position2: Objective Plan-Apochromat 20x / NA 1.0 W DIC (WD=1.8mm)

-Position3: Objective Plan-Apochromat 20x / NA 0.8 Air

-Position4: Objective Plan-Apochromat 40x/1.4 Oil DIC (WD=0.13 mm)

-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

Detectors:

Two very sensitive Gallium arsenide phosphide photomultiplier tube (GaAsP PMTs) detectors

with free choice of the spectral range. (Uses a tunable short pass and band pass filter in front

of the PMTs)

Be very careful with using it to prevent instrument damage! If oversaturated, detectors will

switch off automatically.

1 GaASP PMT for the Airyscan detector that can function as a normal detector in confocal

mode (detection range adjustable by a tunable short pass and a tunable long pass filter, no

bandpass filters)

An ESID detector for transmitted light.

Simultaneous detection of 3 dyes possible.

VII. Starting up:

1. Switch on the multiple socket (1) “Hanging on the right side of the rack”. 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” -> Restart the ZEN if it is open!

(preventing bugs and accidental objective crash)S

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

VIII. 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

8. Before switching to acquisition mode, close transmitted and reflected light shutters by

pressing ‘off’ and turn off the Cool LED by pressing ON/Off button

IX. Acquiring confocal images

•Select the ‘Acquisition’ tab in the main menu of Zen blue

•Go to “Smart Setup” choose your dyes (set the LUT) and configuration, you can choose

between simultaneous (fastest), Best compromise line scanning mode (Smart) and sequential

(best signal) scan, press ”Apply”. (Avoid Line mode as it blocks the changing of filters and

sliders, therefore any change will affect all the track!)

•Make sure that Red and FarRed dyes are not set to the 1st detector (due to the Variable

dichroic arrangement)

•If you are using the detector for transmitted light (ESID): activate (ESID) represented in the

light path scheme. Adjust intensity in the ESID Gain, then adjust digital offset and digital gain

of the ESID detector in order to “stretch” the histogram and use the whole dynamic range of

the detector.

•

Make sure Köhler illumination is set properly if you intend to use the ESID detector

(for bright field images)!

•Press the “AF” (Autofocus) button to find your focus, alternatively go to “Live” acquisition

modus and find it manually

•Set pinhole to Airy Unit (AU) 1 in the channel dialog to achieve confocality. Note, that activating

the ‘1AU’ button by clicking on it once (gets light blue background color) fixes the pinhole to

1AU. In this case, the match pinhole function in the Z-stack setup will not change pinhole sizes.

•In the ‘Channels’ dialog you can adjust the pinhole, gain and laser power manually

•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.

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

overexposed. If the resulting gain is below 400 V, decrease the laser power. If the gain is above

900 V, increase the laser power.

•To zoom in your image, go to ‘Acquisition mode’ and adjust the slider accordingly. With “Crop”,

you can select the desired area of your image, then press “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’tab to adjust optimal number

of pixels and the pixel size according to Nyquist sampling criteria.

Note: More pixel than suggested by the software as ‘optimal’ rarely make sense. On the other

hand, feel free to decrease the pixel number to increase acquisition speed at the cost of

resolution.

•To capture single images, click on ‘Snap’ (Snapped images are overwritten if you press Live

before saving them)

1. Setting up Z-stack:

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

Press “Live” and set your first and last slice

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

Activate the ‘Optimize sectioning and step’ checkbox to see slice overlapping. Here you can

again set an optimal interval to ensure ~50% overlap between sections.

If you wish to use the match pinhole function, make sure that the ‘1AU’ button in the

‘Channels’ menu is deactivated on all tracks (no blue background of button). This function will

match pinhole sizes to the optimal pinhole size of the lowest wavelength of your imaging setup

and ensure that all sections have the same thickness.

Press “Start experiment”

2. Setting up time series:

Enable “time series”

Time series dialog will open

Set cycle number and interval time

Press “start experiment”

3. 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.

You can also add multipositions in this tab:

Important: to make sure that the software images the positions in the order you added them,

make sure that you DISABLE the feature “tile regions/positions” in the options menu of the

Tiles bar (see picture on the next page)

4. Data Saving:

Save your data to D: data folder. Note that this computer is not backed up. Please move your

data as soon as to the file server, all the user data will be deleted automatically after one

month.

Disable this option to keep

the order of positions during

imaging!

X. Switching off:

1. DO NOT save any “Experiment” or overwrite the default “Experiment” (marked with a home

logo) as this setting will be used in the next startup 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. 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 coveringsor 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) as well 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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