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 Histochemistry 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. Fill in the log book (‘no bulb’) 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 ‘RGB’. 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 ‘colour’ 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. Depress 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. 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. The light adjustments are located on the right-hand side of the microscope. You can turn the light on and off by pressing this button The light intensity is controlled by the black dial next to the main on/off switch. For histochemisty images it is recommended you use the light at it’s highest intensity. Press the 3200k button to set the light at the correct intensity for imaging.

If the image down the eyepieces has a strange colour to it, the filter wheel may not have been replaced to it’s proper position. Change the filter wheel using the directional buttons located under the focus wheel on the left-hand side of the microscope until ‘blank’ is displayed on the black disk underneath the eyepieces. 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 to a non-oil objective, make sure you clean the oil off of your slide first. 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. You must also remember to clean the oil off of the oil-lens using the lens tissue provided. 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/histology Select the channel 4. 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.

Next you need to adjust the white balance. Ensure the ROI tool is selected and use it to mark an area of your image which should be pure white (i.e. either lacking in sample or a piece of white paper can be used if preferred). Then go to ‘video’ and select ‘auto white balance’. It will take a short time for this to be calculated, be patient. You can adjust the colour balance manually by using the individual colour slider bars. 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.

Brilliant Image If you are taking multiple images of the same sample it is possible to name the images before you have taken them. Double click on the acquisition set up icon The acquisition set up window will open. At the very top of this window, fill in the ‘title’ box with your sample identity and click ‘OK’. Every image taken will now be labelled with the wording in the title window with numbered increments (1, 2, 3 etc). When you wish to return to viewing down the eye pieces, simply push the silver lever on the right-hand side of the microscope back in.

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 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 microscope and computer monitor. Replace dustcover over 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!