Zeiss AxioObserver Manual de usuario

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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

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 »

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.

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