REMEMBER: You have 5GB of disk space on this microscope. Check before you start if you have room for your experiment. If not delete your old data.

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1 1 Use of the Zeiss LSM 510 Inverted 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 immediately. REMEMBER: You have 5GB 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 1. Start the software Click the ZEN shortcut button Click start system

2 The screen looks like this once you have switched everything on. The control menu is on the left and you work from top to bottom. Your images are stored on the right. Light path (eyes and laser is controlled here) 2 2. Turn on the Lasers To turn on lasers select LASER menu, RHS of workspace. Drop it down using the small arrow in the left corner. Select the lasers you need for your experiment and turn them on For the Argon and Enterprise lasers drop down the Laser Properties arrow For the Argon laser: switch it onto standby, wait until it has warmed up and then switch on, and set output to 6.1A. NB is this uses more than 50% of the laser output see Ann/Amanda For the Enterprise laser: switch it onto standby, wait until it has warmed up then switch on, and set the output to 100%

3 3 3. Select the Configuration you need Pick Imaging Configuration from the drop down menu Select the configuration you need Click the icon to activate your chosen configuration 4. Set the Microscope up and find your specimen Click the Ocular button to send the light to your eyes (top right area of the workspace) This will allow you to check your samples down the microscope before you start laser scanning In the Light Path control menu: This opens the transmitted (Brightfield) light Select the objective lens that you want to use Select the appropriate filter (DAPI/FITC/TRITC) Click the fluorescence shutter Open to see fluorescent light

4 4 5. Start confocal scanning Click LSM button (top right of the workspace) Click the Fast button (found on top left of the workspace) your Click Split on the left hand side of image (this splits the image according to channels used) 6. Set the levels for each channel Click the coloured region under each channel (in the panel underneath the image window) to activate the range indicator palette; the images will now go grey or Red and Blue Make sure the channels menu is in your workspace, if not click the right hand side arrow to 'float' the menu out For each channel and change the following in each to obtain good image: 1) Pinhole : click on 1 to make pinhole 1 airy unit (Please note: if you re doing colocalisation, refer the worksheet on the BALM website) 2) Master 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

5 5 7.Resize your images using the crop tool. To stop scanning at any time click Stop Stop scanning and press Crop (bottom of screen) Simply put the cropping window (Red box with cross hair) over the region you want to crop Change the position, size or rotation of the cropped area by dragging the edges of the crop box with the cursor Start scanning again and your cropped image will appear 8. Set up the scanning parameters and selecting the area you want to image Once you are happy with your image stop scanning Go to the Acquisition Mode tab on the LHS of the workspace and float out if not in workspace already. Generally for a nice detailed picture: Frame size should be 1024x1024 Scan speed: 7 Data depth should be 16 bit Scan average should be 4 Optional if you don't want to use crop To alter the scan area drop down the arrow While the image is scanning: You can now expand or reduce the scan area, Zoom in or turn it around as required

6 6 9. Take the picture Click Single in the scan menu 10. Saving your Image(s) Go to the Open Images section on the top right of the workspace Select the one that you want to save Click the floppy disk icon to save the image (please save to your folder in Users on E drive!!) To convert to tif go to the file menu (top right) and click Export. To export just the raw data that you have pick the format (.tif) click export Raw data Single plane (normal scan) or Series (Z stack or timelapse) To export the image display including any overlays or analysis that has choose export Contents of Image window or Full resolution image window Single plane (normal scan) or Series (Z stack or timelapse). Choose 'select file name and save' to save

7 7 12. To take a Z stack Go to the Z Stack toolbar (found on the left side of the workspace) and drop it down Click on the tick to activate the Z stack menu (you can't collect a z stack without this) Its white when activated Choose if you want to select first and last planes or to collect data from a central point (BALM staff recommend select first and last in general) For stacks collected in select First and Last mode Enter your required interval for z stack Select Keep interval Note: use Optimal Interval if you want the best resolved stack (for 3D image generation) Click Fast to start scanning Move to the bottom your sample and hit set last Move to the top of your sample and click set first Click START to collect the Z stack You can view the z series (and time series) as it is being collected using the Gallery option 13. Collecting a time series / timelapse Go to the Time Series icon and drop it down. Make sure you activate the tick (Right hand side) Select the time interval and how many image you can to collect. Click START to start the timelapse NB an icon of a pile will appear below start when a Z series is being collected and a icon of a stopwatch when a time series is being collected