Use of the Zeiss LSM 510 Meta

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Use of the Zeiss LSM 510 Meta
Firstly please be aware that this microscope should be treated with respect and care at all times.
Rules of use:
This Microscope can only be used by Masters by Research or PhD students, Postdocs and members of staff.
You must receive training from Ann Wheeler before you use this microscope..
The microscope lenses must be cleaned after every usage and the equipment treated carefully at all times.
If you have any problems at all with the microscope, no matter how trivial they may seem please see Ann
Wheeler, Amanda Wilson (or in an emergency Gary Warnes) immediately.
REMEMBER: You have 2GB of disk space on this microscope. Check before you start if you have room for your
experiment. If not delete your old data.
To switch on the microscope.
Switch on the Mercury arc bulb
Switch on the remote control button
Switch on the computer
Clean the objective lens you want to use before you start work
Login to the computer.
Open the software by hitting AIM software shortcut button
Choose SCAN new images and EXPERT mode from the menu.

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To take a picture:
First you need to set up a folder to store your images. Go to File and select the New icon.
Set the location of the database to be in the E: drive in your user area
Name your image database.
o Danger if you do not have a database the images you take will not be stored.
1. Switch on lasers.
Go to Acquire menu and Select Laser (fig 1)
the laser menu will appear (fig 2)
Switch on the lasers needed for your experiment
Fig. 1 Acquire menu
Fig. 2 Menu for turning on lasers
Argon laser: switch it onto standby,
wait until it has warmed up and switch on.
Set output to 6.1A.
Fig. 3 Menu for configuration
2. Select the Configuration you need
Pick Config from the acquire menu (Fig 1).
Select Multitrack (Fig 3)
‐ click on config button (looks like a disk)
‐ go to drop down menu (Fig 4)
Fig. 4 Drop down menu for multi‐tracking
‐ Select the configuration you need
‐ Hit Apply
‐ Optional If you have custom made a configuration hit Store

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3. Set the Microscope up and find your specimen
Fig 5. Microscope control menu
‐ Select Micro from the Acquire menu.
The microscope control menu will appear
(Fig 5.)
In the microscope control menu:
Select the objective you want
Select the different objective lens and filters
using reflector (red/blue/green)
Click reflected light to open the fluorescence
shutter
4. Start collecting confocal images –
Pull slider the right hand side of the microscope fully out.
Select Scan from the menu.
Fig 6. Part of the
scan control menu
The scan control menu will appear (Fig 6)
‐ Pick Fast XY
‐ Go to channel and adjust the following ‐
zoom: 1, rotation: 0, offset: middle
‐ Select Split XY (splits the image according to wavelength)
‐ On the image menu (fig 9), click palette

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‐ Select range indicator
‐ The image will now go grey or Red and Blue
(You want your image to be grey ideally with no red and blue)
5. Align the laser light to the Photomultiplier tubes (detectors)
In the Scan menu there are two menus :
Mode and Channel ‐ Select Channel (Fig 7)
Fig 7. Scan control toolbar – Channels menu
To align the detector go into the different channels
(red/green/blue) and change the following in each to
obtain good image:
1) Pinhole : click on 1 to make pinhole 1 airy unit
2) Detector gain: increase level until you see red, then
reduce again until red just disappears
3) Offset: decrease level until you see blue, then increase
again until blue just disappears
Once image in all channels is fine click STOP
6. Set up the scanning parameters
Now select Mode in scan toolbar and check parameters:
Generally for a nice detailed picture:
Fig 8. Scan control toolbar – Mode menu
‐ Frame size should be 1024x1024
‐ Scan speed: 7
‐ Data depth should be 12 bit
‐ Scan average should be 4

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7. Take the picture
Click Single in the scan menu
1. To get your RGB image back,
‐ Click Palette in the image menu (fig 9)
‐ Select No palette (to obtain colour image)
‐ Close palette menu
To save your image hit Save right hand side of your image
‐ The image is now saved in the database you created at the beginning of your session
If you want the RGB merge image go to the menu on the left hand side of your image and choose xy
To convert image to tif. Go to File on the acquire menu (Fig 1).
Go to Export, select tif and save in LSM4 format
Choose ‘contents of image window’ from the drop down menu.
Fig 9. Image menu
7. To take a z stack
1. Check z stack on the scan control toolbar (Fig 7 and 8)
Enter your required interval for z stack
Select Keep interval.
Start scanning using Fast XY scan
Move to the top of the z stack and click mark first
Move to the bottom of the z stack and hit mark last
Check scan speed in Mode menu
Hit start