Zeiss LSM 710 User manual

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6

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NeuroTechnology Studio

Zeiss LSM 710

User Manual

For questions or concerns contact:

Lai Ding: 781.901.6116 or lding@bwh.harvard.edu

2

TABLE OF CONTENTS

Startup

3

Opening ZEN

7

Channel Setup

8

Live Mode

9

Z-Stack

10

Shutdown

11

3

Startup

4

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.

5

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.

6

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!

7

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.

8

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

9

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

10

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.