Zeiss AxioObserver User manual

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Switch on the temperature controller.................................................2
Switch on the CO2 controller ..............................................................3
Start the system ..................................................................................4
Equipment...........................................................................................5
Microscope stand presentation ..........................................................6
Start Zen / Observation with the oculars ............................................8
Acquire a widefield image in transmission mode with the color
camera ................................................................................................9
Acquire an image (transmission/fluorescence) with the black/white
camera ..............................................................................................11
Acquire a Z-stack serie ......................................................................12
Acquire a time serie .......................................................................... 14
Multiple stage positions ....................................................................15
Tile scan ............................................................................................16
Graphic tools .....................................................................................22
How to save your data ......................................................................23
Switch off the system........................................................................24
Switch of the temperature and CO2 system .....................................25
WIDEFIELD ZEISS AXIO OBSERVER
USER MANUAL

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Switch on the temperature controller
It’s important to switch on the temperature at least 30 minutes before the beginning of the
session. Once your sample is put on the system and you saved the positions you wanted to
acquire, to avoid ab important focus shift in z, it’s best to wait at least 1 hour and re-set the
focus on your positions
1. Turn on the temperature controller with the red switch.
2. Choose the desired temperature (displayed in green) with the buttons up (+) and
down (-).
The real temperature is displayed in red.

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Switch on the CO2 controller
1. Open the air compressed bottle (silver pressure regulator) and the CO2 bottle (golden
pressure regulator) in the anti-clockwise direction.
2. Turn on the CO2controller (red brick).
3. Turn the air compressed bottle valve (silver) clockwise until you obtain a pressure of 1 Bar
in the exit of the pressure regulator (right pressure gauge).
4. Take the greenish tube connected to the CO2controller (« MAIN out » exit) and clip the
end of the tube to the water reservoir on the system.
5. Put the CO2cover on the sample holder.

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Start the system
1. Turn on the multi-socket power (on the shelf to the left of the system). The fluorescent
lamp is switched on at the same time.
2. Switch on the microscope by pushing the « START » button on the left side of the system.
3. Switch on the PC, use the « IJM » session.

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Equipment
Motorised inverted microscope Zeiss Axio Observer Zeiss
Black/White camera Axiocam MRm (pixel size 6.45*6.45 µm)
Color camera Axiocam HRc (pixel size 6.45*6.45 µm)
Motorised stage in XY
LED box CoolLED pE-300white for the fluorescence
Phase contrast and DIC available in transmission
Available filters:
excitation
dichroïque
émission
fluo
BP 335 - 383
BS 395
BP 420 - 470
DAPI
BP 424 - 448
BS 455
BP 460 - 500
CFP
BP 450 - 490
BS 495
BP 500 - 550
GFP
BP 538 - 562
BS 570
BP 570 - 640
DsRed
BP 625 - 655
BS 660
BP 665 - 715
CY5
Available objectives:
EC Plan-NEOFLUAR 5x/0,16 Ph1
(420331-9911)
Plan-Neofluar Dry 0,16 0,17 18500 5,2 I 5-1
Plan-Apochromat 10x/0,45 Ph1
(420641-9910)
Plan
Apochromatic
Dry 0,45 0,17 2000 10,3 II 10-1
Plan-Apochromat 20x/0.8
(420650-9901)
Plan
Apochromatic
Dry 0,8 0,17 550 8,2 II 20-1
Sur l'étagère
On the shelf
LD Plan-NEOFLUAR 20x/0,4 Ph2
Korr (421351-9970)
Plan-Neofluar Dry 0,4 0-1,5 7900 (0,75) 2 II 20-2
Sur l'étagère
On the shelf
LD LCI Plan-Apochromat 25x/0.8
Imm Corr DIC (420852-9870)
Plan
Apochromatic
Oil 0,8 0 - 0,17 570 5,2 II 25-1
Plan-Apochromat 40x/1.3 Oil DIC
(420762-9800)
Plan
Apochromatic
Oil 1,3 0,17 210 5,4 III 40-1
Plan-Neofluar 40x/0,6 LD
(421360-9970)
Plan-Neofluar
Dry 0,6 0 - 1,5 2900 1,1 II 40-5
Sur l'étagère
On the shelf
LD Plan-NEOFLUAR 40x/0,6 Ph2
Korr (421361-9970)
Plan-Neofluar Dry 0,6 0 - 1,5 2900 (0,75) 1,1 II 40-6
Sur l'étagère
On the shelf
Plan-Apochromat 63x/1.4 Oil DIC
(420782-9900)
Plan
Apochromatic
Oil 1,4 0,17 190 2,5 III 63-1
Plan-Apochromat 100x/1.4 Oil DIC
(420792-9900)
Plan
Apochromatic
Oil 1,4 0,17 190 1III 100-1
DIC
Number
Objectives
(ref Zeiss)
Type
Immersi
on
Numerical
Aperture
Cover glass
Free Working
Distance (µm)
Relative
brightness

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Microscope stand presentation
Left side
1. Take the objectives down at the lowest position.
2. Take the objective back up to its working position.
3. Open/Close the fluorescence shutter.
4. Filter cube change for fluorescence (2 buttons).
5. Macrometric knob.
6. Micrometric knob.
Right side
1. Take the objectives down at the lowest position.
2. Take the objective back up to its working position.
3. Open/Close the fluorescence shutter.
4. Filter cube change for fluorescence (2 buttons).
5. Macrometric knob.
6. Micrometric knob.
7. Open/Close the brightfield shutter in « TL ».
8. Open/Close the fluorescence shutter in « RL ».
Front side
1. Light intensity adjustment (wrench adjuster).
The Joystick
Move the motorized stage in X and Y.
Hold the joystick button to move fast.
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TFT screen (touch screen)
1. Open/Close the Brightfield shutter.
2. Open/Close the fluorescence shutter.
In « Microscope », in the « Control » menu, there are
several tabs allowing you to:
3. Choose the objective « Objectives ».
4. Choose the filter cube for fluorescence
« Reflector ».
5. Add/Remove a 1.5X lens « Optovar ».
6. Observation in brightfield « BF », phase contrast
« PH » or Normanski contrast « DIC ».
7. Aperture Diaphragm opening (for the köhler
illumination setting)
8. Power of the halogen lamp
The LED for the fluorescence
The fluorescent lamp is composed of 3 LEDs.
1. The first one is for the DAPI
2. The second one is for the GFP
3. The third one is for the mCherry and Cy5.
4. Select the wanted LED « select ».
5. Press the button On/Off to switch on and off the
LEDs.
6. Adjust the power of each LED « +/- ».
7. Turn off the LEDs « On/Off » when you don’t use
them to increase the lifetime of the LEDs.
LEDs on (black) LEDs off (grey)
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Start Zen / Observation with the oculars
1. Start the « ZEN » software (Icon on the desktop).
2. The stage calibration is necessary only if you use
multiwell plates or if you want to retrieve positions
from a past experiment
3. Select the tab « Locate ».
4. « HAL» allows you to observe your sample in brightfield (transmission).
If you want to observe in phase contrast or DIC, first
select « HAL » then select « PH » or « DIC » on the TFT
screen
5. « DAPI » to observe in blue.
6. « CFP » to observe in cyan.
7. « GFP » to observe in green.
8. «DsRed » to observe in orange-red.
9. « Cy5 » to observe in far red.
10. « Light off » close the shutter for the transmission
and the fluorescent light.
For transmitted light acquisitions, phase contrast or DIC, it is important to make adjustments
on the microscope (set the Khöller illumination…)
Refer to the panels in the room of the microscope.
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Acquire a widefield image in transmission mode with the color camera
Acquisition in transmission mode
Before observing in brightfield, you have to set the
Khöller illumination, then if you need to the phase
contrast or DIC (refer to the posters in the microscope
room).
Select « BF », « PH » ou « DIC » on the TFT sreen.
1. Select the tab « Acquisition ».
2. Choose « TL - Camera couleur ».
3. Open the menu « Channels ».
4. Check the line « TL Brightfield ».
5. Make a « Live ».
6. Adjust automatically the exposure time by clicking
on « Set Exposure »
Or
7. Adjust manually the exposure time.
8. Make a « Snap » to acquire your image.
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Adjust the white balance
Adjust the « White Balance » allows to define a white reference in the image that you are
acquiring.
1. Select the tab « Acquisition ».
2. Open the menu « Acquisition Mode ».
3. Make a « Live ».
4. Click on « Pick. . . », a little pipette will appear instead of the mouse arrow.
5. Choose an area that is suppose to be completely white in your image and click on it. The
correction is done automatically.
6. The « Auto » option allows to correct the white balance, but preferably in a region
without any sample.
AFTER
BEFORE
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Acquire an image (transmission/fluorescence) with the black/white camera
Before observing in brightfield, you have to set the
Khöller illumination, then if you need to the phase
contrast or DIC (refer to the posters in the microscope
room).
Select « BF », « PH » ou « DIC » on the TFT screen.
1. Select the tab « Acquisition ».
2. Choose « Fluo + TL - Camera N&B ».
3. Open the menu « Channels ».
4. Check the line(s) with your fluorophores of interest.
5. Select one channel (light grey).
6. Make a « Live ».
7. Set the exposure time automatically by clicking « Set
Exposure »
Or
8. Set manually the exposure time.
9. Do it for every color of interest.
10. Make a « Snap » to image the checked channels.
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Acquire a Z-stack serie
Stack acquisition
1. Check the « Z-Stack » option.
2. Check « Show all Tools ».
3. For multicolor acquisitions, choose the way you
want to switch channels : make the entire z-stack
of one channel, then switch to the other one
« Full Z-Stack per Channel » or all channels per
plane « All Channels per Slices ».
Two acquisition mode exists: you can define the top
and the bottom of your stack, or define only the
center of you stack.
4. In the menu « Z-Stack » Check the tab
« First/Last »
5. Make a « Live ».
6. « Set First » defines one extremity of you stack
and « Set Last » the other one
OR
4. In the menu « Z-Stack » Check the tab « Center ».
5. Make a « Live ».
6. Define the center of your stack with « Center ».
7. « Interval » must be checked to ensure a fixed
interval value.
8. Enter the wanted interval value in µm.
9. Too choose the optimal resolution, you can use
the « Optimal » option.
10. « Range » indicates the thikness of your stack.
11. The number of planes can be changed in « Slices ».
12. Start the acquisition by using « Start Experiment ».
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Display the stack
Your stack is displayed in the ZEN interface at the end of the
acquisition.
In the tab « Dimensions » :
13. « Z-Position » allows you to see the different planes of the
stack.
14. « Gallery » displays a set of all the images composing the
stack.
15. « Ortho » allows an orthogonal view of your stack.
16. « 3D » allows a 3D reconstruction of your stack.
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Acquire a time serie
1. Check the « Time Series » option.
2. Open the menu « Time Series ».
3. Define the time interval between two
images.
4. Choose the number of cycles.
5. warning: The time the microscope make to
acquire a z-stack (or tile) has to be taken in
account in the chosen interval. You can
measure it by clicking on « Measure Speed»,
the value will be shown in the « Interval »
rubric
6. You can minimize the time interval by
checking « Use Camera Streaming if
Possible ».
Your time serie is displayed in the Zen interface
at the end of the acquisition.
7. In the « Dimensions » tab you can look a the
different time point of your acquisition
« Time »
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Multiple stage positions
To make the software take in account the z
coordinate for each position you have to :
1. Open the menu « Focus Strategy ».
2. Choose the option « Local Focus Surface ».
3. Choose the option « Fixed Z-position ».
4. Check « Tiles ».
5. Open the menu « Tiles ».
6. Open the rubric « Positions ».
7. Click on « Advanced Setup » to display the navigation space that will allow you to set
your positions.
8. Make a « Live ».
9. On the navigation space, double click where you want to acquire an image, or use the
joystick to move the stage.
10. Once positioned, click on the arrow next to the « Positions » rubric.
Repeat this operation every time you want to save a new position.
11. In the tab « Single Positions », you can see all the positions that you have saved.
12. Click on « Start Experiment ».
It’s possible to use different format of well-plates. Don’t hesitate to ask to the imaging
facility engineers for advices.
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Tile scan
Tile scan acquisition
1. Check « Tiles »..
2. Open the menu « Focus Strategy ».
3. Choose the option « Local Focus Surface ».
4. Select « Fixed Z-position ».
5. Open the menu « Tiles ».
6. Click on « Advanced Setup ».
7. Make a « Live ».
8. The navigation space is displayed in the center
of the software interface. Double click where
you want to acquire an image, or use the
joystick to move the stage
9. In the menu « Tile Regions », click on « Tiles »
and enter the dimension of the desired tile.
Your current position will correspond to the
center of the tile.
10. Click on « + », the tile is then displayed on the
navigation space.
11. Click on « Star Experiment » to start the
acquisition.
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Focus correction
There are two ways to correct the focus on the entire
tile. It has to be done before any acquisition.
12. Click on « Support Points » below the navigation
space.
13. Enter the number of positions on wich you wish to
correct the focus.
14. Click on « Distribute ».
15. Yellow circles will be distributed in the tile.
16. Double click on a yellow circle, make a « Live », set
the focus and click on « Set Current Z » to save it.
Repeat for each yellow circle.
OR
17. Double click on the position where you wish to
correct the focus. Adjust the focus.
18. Click on « Add Support Point at Current Stage and
Focus position», a yellow circle will appear.
19. Repeat the process on at least 5 positions.
20. Click « Start Experiment » to start the acquisition.
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Online shading correction
During you are adjusting your acquisition parameters, you can correct the shadow effects
online.
Find a position without your sample
1. In the channels toolbox:
2. select the “global” correction mode in the list.
3. Click on “Define”
4. The shading option is automatically selected.
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Tile scan finalization in fluorescence and transmission during the acquisition
With this option : stitching of tiles is done during the acquisition
In the “Tiles” toolbox and in the “options” section:
1. Select « Stitching During Acquisition »
2. In the section « Stage Travel Optimization »
select « Comb »
3. Set the « Tile Overlap » at 20%
4. Select “Image pyramid during acquisition”.
Thanks to this option, an image pyramid is
generated during the acquisition. This optimizes
the image for fast display.
If this option gives bad results, it is better to finalize the
tile scan after the acquisition (see below)
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Tile scan finalization in fluorescence after the acquisition
For this option, the checkbox “online stitching” has to be unselected during the acquisition
(see above).
1. Click on the tab « Processing ».
2. Click on « Single ».
3. Open the menu « Method ».
4. Go to the « Geometric » rubric and select « Stiching ».
5. Open the menu « Input » and select the tile you want to
stitch.
6. Open the menu « Parameters ».
7. Click on « New Output ».
8. Check « Fuse Tiles ».
9. If the shading is not correct, click on « Correct Shading » and
select the « Automatic » mode.
If you have several colors (channels) or a z-stack :
10. Open the menu « Select dimension reference for stitching ».
11. Click on « All by reference » and select a channel that will be
considered as the reference.
12. Choose the z that will be considered the reference.
13. Click on « Apply ».
14. The adjusted image is called « IP- Stitching ».
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