Zeiss LSM 880 User manual

880 Table of Contents
880 User guide overview………………………………………………...2-9
Setting up configurations on your own…………………………...10-11
Time Series………………………………………………………………………15
Z-Stacks……………………………………………………………………………16-17
Calibration for Airyscan…………………………………………………….22-25
Acquisition mode-Recommended settings………………........12
Tile Scans and Stitching…………………………………………………...13-14
Adding a DIC Transmitted Light Image……………………………..18
Kohler Illumination…………………………………………………………..19
Incubation………………………………………………………………………..20-21
1

880 User Guide Overview
• Starting up the system
• Turning on the lasers
• Viewing your sample
• Setting up a configuration
• Taking an image
• Saving your file
2

Starting up the system
1. Follow the directions for Powering up the 880, this is located on the wall behind the
880 monitor
2. Double click on the Zen icon
3. Click on the Start System tab
3

Turning on the lasers
1. The system starts with the locate tab open
2. To turn on the lasers click the acquisition tab
3. Use the pulldown tab to turn on the lasers
Note: the 488 laser takes about 5
minutes to warm up.
4

Viewing your sample
1. To view your sample through the eyepiece
click the locate tab
2. Choose your objective by clicking on the objective
button, this will bring up a list of objectives
currently on the microscope.
You may also choose your objective by using the
touch screen next to the monitor – Touch “Home”
then “Microscope” and click on the “Objectives”
Tab.
3. After you choose your objective make sure to
clean it with lens cleaner
4. Set your sample on the stage, please remember
to add oil or water as appropriate
5

Viewing your sample
1. To view your sample through the eyepiece
select what color you would like to use.
If you would like to see white light select DIC
Blue Green Red
2. Use the focus wheels on the side of the
Microscope, or on the side of the touch
screen to bring your sample in focus.
Towards yourself moves the objective down
Away from yourself moves the objective up
3. Use the joystick next to the microscope to move your sample.
6

Setting up a configuration
1. To load a configuration from a previous experiment, go to
file, open, then choose the experiment you would like to
reuse
2. Click the reuse button either on the top left of the screen
or at the bottom of your image
3. Please contact Jeff, Agnes or Erica if you need to set up a new configuration.
7

Taking an image
1. To view your sample click on the Live button.
2. You may need to adjust to focus by using
the focus wheels on the microscope or on the
side of the touch screen.
3. To change the intensity of your sample go to
the channels tab and click on the track you
want to adjust. You can increase or decrease
your intensity by adjusting the Gain(Master)
It is recommended you stay between 600-800
4. You may also need to adjust the laser power.
Please talk to Jeff, Agnes or Erica if you have
questions about selecting the appropriate level
for the laser intensity.
4. Once you have your image in focus and at the intensity you like, click the
Snap button to acquire your data quality scan. 8

Saving your file
1. After you have taken your image you may save your file by using one of the following
options:
-clicking on File, then Save
-clicking on the disk icon on the top left
-clicking on the disk icon on the right, under images and documents
9

Setting up configurations on your own
1. Click on button that looks like an open file folder
it will bring up a menu called
Load Acquisition Parameters
2. Choose what laser lines you would like to use
to excite your sample. Then click on the setup
that contains those lasers.
Note: Examples of commonly used dyes and the
laser lines used to excite them are listed on the
next page.
Note: The files that start 880 are for the regular
confocal and the ones that start AS are for the
Airyscan
10

What laser should I use?
Here are some examples of commonly used dyes and the laser lines used to excite them:
Laser Dye
405 Dapi, Alexa 405
458 CFP
488 GFP, FITC, Alexa 488
514 YFP
561 Alexa 543, Alexa 568, Alexa 594, Rhodamine,
Mitotracker red
594 Alexa 594
633 CY5, Alexa 633, Alexa 647
11

Acquisition mode – Recommended settings
1. A good Frame Size to use is 512x 512, if you need
to change your Frame Size click on the X*Y button
2. Speed should be set about 7, if you just want
to do a quick scan you can increase the speed
by clicking on Max
3. For Averaging 2 or 4 are a good numbers to use
4. Bit Depth should be 8 bit
5. Direction: you can scan faster by using
bidirectional scanning <-->
6. If you would like to see more detail in an area
you can increase the zoom
12

Tile Scans
1. To do a tile scan, first click Tile Scan, a Tile Scan box
should appear on the right if it doesn’t scroll down on
the left until you see it listed under Multidimensional
Acquisition.
2. If you would like to move the tile scan box, click
the undock button
3. Under the Centered grid tab choose the
number of Horizontal and Vertical tiles you
would like to use.
4. Enter an overlap of 10%
5. Click the start experiment tab
13

,
Stitching a Tile Scan
1. After you take your image save your file
-click on File then Save or
on the top left-click on the disk icon
2. Click on the Processing tab
3. Under Method click on Stitch
4. Click on the image you would like to stitch
and then under Method Parameters click on Select
5. Next click on Apply, this will stitch your image for you
6. Finally save your file, it will have the original image name with an “_Stitch” at the end.
14

Time Series
1. To do a time series, first click on Time Series.
2. The Time Series tab will appear under the
Multidimensional Acquisitions on the left side.
To move the Time Series box , click the
undock tab
3. Set the number of Cycles for how many images
you would like to take.
4. Set the Interval for how often you would like to
take an image.
5. Click Start Experiment
15

Z-Stacks
1. To make a z-stack first click on Z-Stack, a Z-Stack
box should appear on the right if it doesn’t
scroll down on the left until you see it.
2. If you would like to move the Z-stack box, click
the undock button
3. Click on live
4. Find the bottom of the sample, roll the focus wheel
towards your body, then click Set First
5. Find the top of the sample roll the focus wheel
away from your body, then click Set Last
6. Set your interval and check your number
of slices **note if you are doing 3D you
must oversample, check with us for your interval
7. Click on start experiment 16

4. Click on Center, find the center of the sample you
6. Set your interval and check your number of slices
Z-Stacks using Center
1. To make a z-stack first click on Z-Stack, a Z-Stack
box should appear on the right if it doesn’t
scroll down on the left until you see it.
2. If you would like to move the Z-stack box, click
17
the undock button
3. Click on live
would like to image. Click on the C to take a Snap
of the center image to make sure you are in the
right spot. Click Center to confirm this is the slice
in the middle of the stack.
Make sure you have an odd number of slices
**note if you are doing 3D you must oversample,
check with us for your interval
7. Click on start experiment

Adding a DIC Transmitted Light Image
1. If you would like to add a DIC transmitted
light image click the T-PMT while you are
under the Acquisition tab.
2. To have the best DIC image check your
Kohler illumination, if you need help with
this ask Jeff, Agnes or Erica or check out the
Kohler illumination cheat sheet on the
next page.
18

Kohler Illumination for the 880
1. Bring the sample into focus
2. Close the field diaphragm until
you can see at least one edge
3. Adjust the condenser height
until the Edges of the diaphragm
image are crisp
4. Center the diaphragm image using the
two centering screws
5. Open the field diaphragm, just until
the image fills the field of view
19

Incubation
1. Before you plan to use incubation please talk with Jeff, Agnes or Erica to help with setup
2. Make sure to turn on the incubation and CO2 controls before you open Zen (there is one
switch on the TempModuleS and one on the Heating Unit XL S)
Note: The TempModuleS light will turn on when you switch it on, the light on the
Heating Unit XLS will not turn on until the temperature is turned on in Zen
3. Under the Locate tab you will find the Incubation tab
4. Check the H Insert P if you would like to use the
heated insert, check the H Unit XL if you would
like to use the incubation chamber.
Note: Once you check the H Unit XL the bottom
light on the Heating Unit XL S will turn on and the
middle light will blink yellow telling you the
heating unit is warming up
20