Mercury bulb Camera Microscope on/off Stage up/down Focus wheel Objective controls Fluorescence filter controls Mono/RGB selection lever Microscope/computer Selection lever Light on/off Fluorescence filter info Condensor turret disk Stage positioner

Using the CCD microscope for Fluorescence You should not use this microscope unless you have completed training with Rachel Gregory or Liz Brooks. 1.Start up procedure Switch on the computer. Turn on the mercury bulb, and fill in the log book. Turn on the microscope itself via the green switch on the right-hand side. Turn on the camera situated on top of the microscope, you should be able to see the function lights turn on if you hold your palm above it. It is essential that the camera be the last thing to be turned on. Power fluctuations caused by the other equipment may damage the camera! Below the camera there is a black circular turret with ‘RGB’ and ‘Mono’ written on the left-hand side. Ensure the silver lever underneath is turned to ‘Mono’. Also, ensure the microscope/computer selection lever is fully inserted. You are now ready to start the software. On the desktop you should see an icon for a shortcut to Volocity, double click on it. Fill in username and password. If you do not have your own username and password you should not be using the equipment on your own-contact Rachel Gregory or Liz Brooks to book a session. (if you have forgotten your password we can create you a new one-no expense!) Create a new library and label it accordingly for your samples, save this in your user folder.

In the toolbar of Volocity go to ‘window’ and scroll down to select ‘show video preview’. In the control panel that appears on the right-hand side, ensure ‘Monochrome’ is selected from the drop down menu. The microscope is now set for your imaging.

2. Microscope Controls Firstly you will need to select an objective to use. The CCD microscope has 6 different objectives; The x5 x10 and x20 are low power objectives and are therefore good for imaging areas of cells. These objectives do not require oil. The x10 uses Ph1 and the x20 uses Ph2. The x40 and x100 are high power objectives and are therefore useful for imaging single cells or structures within cells. These objectives do require oil. Only the oil provided (518 F) should be used. Both these objectives require Ph3. Place your slide onto the stage. It should slot between the metal clips, don’t push it under the objective just yet! First ensure there is adequate clearance between the slide and the objective. If there is not, move the stage down using the focus wheel. If the stage moves quickly when turning the focus wheels, you will need to change the setting.. The focus wheel has two settings; coarse and fine. We recommend you always use the fine setting. To switch between coarse and fine, there is a button on the left-hand side of the microscope, behind the focus wheel labelled coarse/fine. Press it to switch between the two settings. Using the fine setting will make it easier for you to focus on your sample and will also help prevent any damage occurring to the microscope and/or your slide.

If you are using a non-oil objective, position your sample on the slide underneath the objective. If you are using an oil objective, put a drop of oil on your slide. You don’t need much! It’s now best if you bring the stage down to it’s lowest position. To do this press the ‘down’ button located towards the front of the microscope base on the right-hand side. The stage will automatically move down. Now position your slide so it is directly underneath the objective. Bring the stage up again by pressing the ‘up’ button in the same location. The stage will automatically move up to it’s original position. Positioning your slide under the objective this way allows a clean connection between the objective and the oil, and reduces the occurrence of air bubbles in the oil. We recommend that you focus on your sample initially using light. The light adjustments are located on the right-hand side of the microscope. You can turn the light on and off by depressing this button The light intensity is controlled by the small black dial next to the main on/off switch.

To check your fluorescence, press the light on/off switch. This will toggle you between light and fluorescence. (You may need to press this twice the first time you switch over to fluorescence). You will now need to change the filter for your specific fluorescence. The filters are changed using the directional buttons located under the focus wheel on the left-hand side of the microscope. As you change the filters you’ll notice numbers appearing underneath the eyepieces. 01=blue 15=red 10=green Blank=no filter (for T-light) If you want to go back from fluorescence to light. Change the filter round to ‘blank’ and press the light on/off switch. NOTE: At the end of your session the blank filter should always be left in place. If you decide you want to change the objective you’re using, first bring the stage down to it’s lowered position. Move round to the objective you now wish to use before bringing the stage back up. IMPORTANT NOTE WHEN CHANGING OBJECTIVES! It is imperative that no oil touches a non-oil lens. If you are using oil and want to change the objective, make sure you clean the oil off of your slide first. You must remember to clean the oil off of all the oil-lens used with the lens tissue provided. Also check which Ph setting the objective you are using requires (it will be written on the objective itself. This can affect the quality of the images you are capturing When moving onto a new slide, bring the stage down to its lowered position before changing the slides. This will prevent any damage being caused to the objectives.

3. Viewing your sample on the computer When you have identified an area you wish to image, pull the silver lever located on the right-hand side of the microscope out one stop. This will carry the image from the microscope to the computer. The computer imaging controls are found on the right-hand side of the viewing window. There are 4 channels set up for you to use; FITC/GFP Rhodamine/RFP DAPI/CFP white light/phase Select the channel you wish to image by left-clicking on the appropriate channel. Select ‘auto-exposure (AE)’, the computer will calculate the exposure time for your image. You can alter this by moving the sliding bar or changing the numbers in the panel. Once you have the settings right, right-click on the channel button and select ‘overwrite’. This will save your exposure settings for that channel. To capture a snapshot: Just click on the camera button. The image on the screen will be automatically saved to your library, you can alter its name here by double clicking on it.

4. Imaging multiple channels/colours If you are imaging multiple colours/channels, you can set up the image acquisition. First set the auto-exposure settings for each of the channels you want to image, and overwrite the channels to save your settings. Next you need to set up the image acquisition-double click on the icon. The image acquisition setup window will open up. Add or take away the number of channels you want to image using the +/- buttons in the light path section. Change the colour you are imaging using the dropdown menus for each channel. (1=green, 2=red, 3=blue, 4=white). When you have set the channels, click ‘OK’. To take the image click the record button. Used when capturing Z stacks. For normal Imaging, ensure only 1 slice is selected Used for multiple sites. For normal imaging ensure ‘don’t auto focus’ is selected Used when capturing movies. For normal imaging, ensure only 1 timepoint is selected This allows you to name your image before you’ve taken it. Subsequent images will be named the same with numbered increments Brilliant image!

The image sequence will take a short time to complete, so do not change the microscope positioning until it has finished. When taken, the sequence will be saved in your library. To view the image, double click on it. You can then toggle between ‘image sequence’ to view the individual channels, and ‘image’ to see the channels merged. To save a copy of the merged image, when viewing the image go to ‘Actions’, then select ‘capture’ and ‘snapshot’. A snap shot of your merged image will now be saved within your library. When you wish to return to viewing down the eyepieces, simply push the silver lever on the right-hand side of the microscope back in fully.

5. Shutdown procedure When you have finished with the microscope you must: If you have been using oil, clean oil off of the lens you’ve been using with the lens tissue provided. Move the objective turret round until the 5x objective is in place. Ensure the filter wheel is set on blank. Ensure the stage is left in the ‘up’ position. Fully insert the computer/microscope lever. Unless you have been asked to leave the microscope on, or the next user is waiting you must proceed with the rest of the shut down procedure. Firstly, it is essential the camera is turned off before any other equipment. Turn off the mercury bulb, fill in your usage in the log book. Turn off the microscope and computer monitor. Replace the dustcover over the microscope. Please leave the computer on to allow remote access to Volocity elsewhere in the building. Don’t forget to return the room key to the log book on the 4 th floor!