Zeiss LSM 710 User manual

ZEISS LSM 710 CONFOCAL MICROSCOPE

USER MANUAL

START THE SYSTEM...............................................................................................2

START ZEN SOFTWARE ..........................................................................................3

SET THE TEMPERATURE AND THE CO2CONTROLLERS ..............................................4

OBSERVATION AT OCULARS ..................................................................................5

STATIF PRESENTATION..........................................................................................6

ACQUIRE ONE OR SEVERAL STAININGS ..................................................................7

SAMPLING (IMAGE RESOLUTION) ........................................................................13

ACQUIRE A Z STACK ............................................................................................14

ACQUIRE A TIME SERIES......................................................................................16

ACQUIRE A MOSAIC IMAGE.................................................................................17

ACQUIRE MULTIPLE STAGE POSITIONS.................................................................19

VISUALIZATION OF YOUR ACQUIRED IMAGES.......................................................20

SIGNAL QUANTIFICATION ...................................................................................21

SAVE YOUR IMAGES ...........................................................................................22

SWITCH OFF THE SYSTEM....................................................................................23

START THE SYSTEM

1. Switch on « MAIN SWITCH » button.

2. Switch on « SYSTEM PC » button.

3. Switch on the PC and log in « USER » session.

4. Once Windows session starts, switch on « COMPONENT » button.

5. Light on the fluorescent lamp.

On the power supply box of Argon laser:

6. Check if the button is at the « ON » position.

7. Turn the Power key on « I ».

On the small laser box:

8. Push the button up, on « LASER RUN » position.

9. When the LED is green (5 minutes after step 7) turn the knob around 9 o'clock, avoiding the red LED

lights on.

START ZEN SOFTWARE

1. Start the software by clicking on ZEN 2011 icon on the desktop.

2. Choose START SYSTEM to access to the acquisition menu.

3. Zen software is divided in 3 parts: the left side is dedicated to acquisition parameters, central part is for

displaying images and toolbar and the right side list acquired images.

SET THE TEMPERATURE AND THE CO2CONTROLLERS

1. Open the INCUBATOR window.

2. Enter the temperature value that you need and tick the box.

3. Open the CO2 bottle.

4. Enter the percentage of CO2 and tick the box.

Warning !!!

At the end of your session, please untick all boxes and close the CO2 bottle.

OPEN

WARNING!

Don't close the air bottle!

OBSERVATION AT OCULARS

1. Choose the 1er tab : LOCATE.

2. Choose the adapted configuration for

fluorescence or transmission.

According to the type of illumination that you

use, you can :

3. Light on/light off/choose the intensity of

the transmission lamp.

4. Open/close the shutter for transmission.

5. Light on/light off/choose the intensity of

the fluorescent lamp.

6. Open/close the shutter for fluorescence.

7. Choose the objective.

8. Change the filter bloc for fluorescence or

choose the ANALYSER position for DIC.

9. Choose the condenser prism for DIC

illumination.

STATIF PRESENTATION

On the left side :

1. Focus knob

2. No fonction

3. No function

4. Optovar 1x

5. Previous fluorescence filter

6. Next fluorescence filter

On the right side :

1. Focus knob

1. Set the objective at the lowest position

to install your sample.

2. Set the objective at its working

position.

3. No function

4. Switch from the objective of high

magnification to a lower one.

5. Switch from the objective of low

magnification to a higher one.

6. Open/close the shutter of transmitted light.

7. Open/close the shutter of fluo lamp.

ACQUIRE ONE OR SEVERAL STAININGS

1. Choose the ACQUISITION tab.

2. Tick SHOW ALL TOOLS.

3. In the LASER toolbox activate the 561

laser if you need it (Power On).

4. Open SMART SETUP.

5. In DYE select the different dyes and their

respective color.

6. Several methods are available :

-FASTEST : allows a simultaneous acquisition ; it is the fastest method but the cross talk from one

color to the next channel could be important.

-BEST SIGNAL : each channel is acquired separately. It is the slowest method but it efficiently avoids

the Cross talk phenomenon.

-BEST COMPROMISE : compromise between speed and reduction of Cross talk

Example : blue and red channels are acquired simultaneously, then the green one is acquired alone.

-LINEAR UNMIXING : use spectral capacity of the system to separate simultaneously the different

dyes. First, you need to acquire sample stained with only one color.

Tricks : These modes are a help to start your configuration. The software does not always make the

smartest choice . You have to check by yourself and then make some modifications.

7. In the LIGHT PATH toolbox, each TRACK

represents a sequence of one or several

colors.

8. Add or remove sequences by clicking on +/-

9. Adapt wavelength detection by moving the

window for each channel.

10. Add or remove channels with +/- for each

sequence.

11. If possible select the same dichroic for

each sequence, in order to speed up the

change from one sequence to the other.

If you don't need to use a dichroic for an

acquisition, you have to choose the

« Plate » filter.

12. Activate the 405 dichroic for each

sequence even if you don't use it.

13. Tick T-PMT to acquire images in

transmission in one of the sequences.

14.

14. In CHANNELS toolbox, adjust the pinhole

at 1 AU (Airy unit) for each channel in

order to get the best resolution to signal

ratio.

15. Adjust the GAIN (MASTER) at 550 for each

channel except for the T-PMT.

16. Acquire in LIVE.

17. Adjust the laser power for each channel.

18. You can increase the (MASTER) GAIN to

decrease the laser power but the signal to

noise ratio will decrease.

19. Don't increase the DIGITAL GAIN except if

the GAIN (MASTER) is really high.

20. Adjust the DIGITAL OFFSET to improve the

image by increasing the signal to noise

ratio.

21. To make all the adjustments, it is

recommanded to use the « Range

Indicator » LUT.

In this mode, saturated pixels are red and

black pixels are blue.

22.

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