Zeiss LSM 710 User manual

ZEISS LSM 710 CONFOCAL MICROSCOPE
USER MANUAL
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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

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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.
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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.

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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.
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OPEN
CLOSE
WARNING!
Don't close the air bottle!

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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.
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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.
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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.
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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.

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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.
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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.
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