Zeiss LSM 710 User manual

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NeuroTechnology Studio
Zeiss LSM 710
User Manual
For questions or concerns contact:
Lai Ding: 781.901.6116 or [e-mail]

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TABLE OF CONTENTS
Startup
3
Opening ZEN
7
Channel Setup
8
Live Mode
9
Z-Stack
10
Shutdown
11

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Startup

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To START the Confocal System:
1) Turn on the Main Switch (1).
NEVER TOUCH the key (***). The key remains in the horizontal position.
Next, turn on switches A and B (SYSTEM/PC and COMPONENTS).
1.b) Turn on switch A (SYSTEM/PC)
Wait until the PC is fully started, showing the login window. then
1.c) Turn on switch B (COMPONENTS).
1.d) Turn on computer.

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2) Now turn on the X-Cite Mercury lamp by
the black switch (2)
Check the calendar for users after you--if
someone will be using the microscope after you,
please LEAVE the X-Cite ON!
3) For the Argon laser power supply,
(LASOS) turn the Laser key to the right, to the
horizontal position. This is the ON position.
Do not touch other switches on the power supply.
If the key is in the vertical position, then it is off.
4) Log in to your account on the computer:
Login to the computer using the lab account. Do not share account password with
anyone outside your lab. Remember to log off when you are finished with your session.

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5) Turn the Argon Laser (LASOS) on:
Wait 5 minutes after turning the power supply key on before
you turn the Argon laser on. (A) Make sure the toggle switch
is flipped up to “run”.
(B)-->To set the laser to 50% power, turn the clockwise until
it lights up red. Then slightly turn the dial counter-clockwise
until it changes from red to green. The light should remain
green while imaging.
6) Next, open the software:
Click on ZEN BLACK.
***Note that the ZEN software can only be opened after following all of the prior steps***
A dialog window will pop up. Click the button: Start System. (Our system runs Zen
2011.)
As the hardware configures, you will hear the microscope parts starting to activate. This
is normal. You are now ready to image! Remember to only save files on the D: drive.
DO NOT save files on the C: drive!
****Before changing objectives press LOAD SAMPLE on the touch-screen. This will
lower the objectives and prevent accidents!

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Opening Zen
1. Once initialized, choose what objective you want using the
microscope display screen and place your slide on the stage
2. Navigate to the ACQUISITION tab and click the SHOW ALL TOOLS
box outlined in red below. Turn on the lasers if they are not already
on. Pressing the “laser properties” dropdown will show the current
status of the lasers. Wait a few minutes for the lasers to reach full
capacity.
3. Use the LOCATE tab to view sample using the eyepieces.

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Channel Set up
Load preset settings. LSM_405_488_561_633 (frame
switching) = basic 4 channel
Select any of these boxes to bring up a
corresponding window
IMAGING SET UP WINDOW
oPress the dropdown to select your dye. The
corresponding excitation graph will appear
above. Edit the horizontal scroll bar to cover the
peak of the line.
oSelect Ch1for DAPI/Hoechst, Ch2 for far-red
dye like CY5, ChS1 for all other dyes
oSelect the T-PMT box to use transmitted light
(brightfield)
o
Acquisition Mode Window
oChange your objective here.
oFrame size: higher number = better resolution,
but longer scan speed
oSpeed: slower speeds = better signal quality,
higher chance of photobleaching
oAveraging: smooth images
oScan area: Can ‘zoom in’on a certain area
Channels Window
oCreate or delete a channel using the + and -
buttons
oPick what laser you want to use
oAdjust laser power: higher intensity = higher
chance of photobleaching
oKeep pinhole at 1 AU (airy unit)
oAdjust gain (brightness): higher gain = stronger
signal and more noise

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Live Mode
1. Once in live mode, find focus of sample. The focus plane of scanning
will be slightly different from the focus plane by eyepiece viewing.
2. The Range Indicator button, circled in red below, can help find focus
and determine exposure. The red pixels in the image indicate signal
saturation, and the blue pixels indicates zero intensity.
CHANNEL WINDOW
oCheck the box for what channel you wish to
view. If you have multiple checked, the
system will scan each one continuously and
make a composite.
oThe channel that is highlighted will be the
editable channel
oAdjust laser power and gain for each
channel

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3. Once you have found focus and adjusted the exposure for each
channel, check the boxes of the channels you wish to capture.
4. Press CAPTURE to take a single image of all the channels.
5. Press START EXPERIMENT if you have a Z-stack or Time-lapse.
6. Use the Min/Max or Best Fit on the display histogram to edit contrast.
7. Press the REUSE (recycle) button on an open image to apply the
settings of that image.
8. Press the CROP button on a still image to select a smaller area of
interest. Pressing LIVE again after this will automatically zoom the
scan area in to that specified area.
Z-Stack
oScroll through the Z to find the intended
scan range. Click “Set First” and “Set
Last” to set the begin and end position
oThe system will automatically provide
the optimized number of steps and Z-
step size. You can change the size of
the stack by changing the number of
steps “Slices”, or the size of the step
“Interval”.
oPress START EXPERIMENT to image a
Z-stack.

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To SHUT DOWN the Confocal System:
When you finish your session, check on the calendar again. If you are the
last user of the day. Go to procedure A. If you are not the last user, go to
procedure B.
Do NOT clean the objective! NTS staff will clean them routinely. We only
ask user who use oil objectives to wipe off excessive oil left on the
objectives.
Procedure A (Shut Down)
1) After imaging, make sure to save and copy your data!
2) Switch off all lasers in “Laser” tab.
3) Shut down ZEN and log off of your account. Now shut down the
computer.
4) Flip the Argon laser power toggle switch to idle (#5A in picture above)
5) Turn the key on the LASOS power supply to the left (counter-
clockwise) and wait 5 minutes for the cooling fan to stop (#3 in picture
above)
6) MAKE SURE LASOS POWER SUPPLY has finished cooling before
shutting off power switches!! (You will hear the fan shut off, or wait
approximately 5 minute)
7) On main control panel, turn off “COMPONENTS”, turn off
“SYSTEM/PC”, turn off the Main Switch (#1)
8) Turn off the X-Cite lamp by using the switch (#2)
9) Make sure to leave the confocal clean and tidy. Throw away any
trash including used Kimwipes, lens paper, etc. Take your slides,
dishes with you and turn off the room lights
Procedure B (Standby)
1) After imaging, make sure to save and copy your data!
2) Shut down ZEN and then log off of your user account.