Standard Operating Procedure (SOP) for Shared Equipment: Spinning Disk Confocal Microscope

Transcription

1 Standard Operating Procedure (SOP) for Shared Equipment: Spinning Disk Confocal Microscope This document is to be used as a supplementary guide and not as a replacement for formal training. DO NOT operate the instrument unless you have had formal training Turning on/off the system: 1. Turn on the power strip on the bottom shelf of the table 2. Turn on the microscope and the epifluorescence lamp 3. Turn on the laser by turning the key switch to the on position. Do this ONLY if you intend to take images. If you want to just check your sample, it is advised that you skip this step to prevent unnecessary wear on the laser 4. To turn off, complete these tasks in reverse, making sure the power strip is turned off last (independent of the laser power)

2 Setting up the software (Volocity): 1. Use your NSTC computer login to log into the computer (computer should be/remain on) 2. If you have not done so already install Volocity onto your account. Once you have done this under your account, you do not need to do so again. a. Open C: Drive>Users>Public>Public Documents>Ernie and follow prompts 3. Open Volocity. You may have to type in the Start menu to find it. You can place it on your desktop if you prefer 4. Create a new library and save under My documents. ALL IMAGES MUST BE SAVED TO COMPUTER AND NOT TO ANY EXTERNAL OR SHARED NETWORK DRIVE. Doing so will cause the computer to crash and may break the hard drive. (Images can be exported after they are captured). 5. Click on Video Preview in the top left to activate certain functionalities 6. On the right side of the window, make sure Auto Contrast is unchecked. If this is checked, the screen will flash constantly as it is constantly adjusting the contrast and is just tough on the eyes 7. On the top right side of the window, double click on the blue 6 and check the requires manual intervention box. This pauses the system just before capturing this channel and allows you to switch light sources. This pause can be added for any channel. 8. From the top left menu, go to Video>Acquisition Setup. Under Channels/Z on the right side, switch Change Focus Using to Zeiss Focus Drive and change Order channels and Z by to Z first then channels. Click OK and open the same menu again. Switch Change Focus Using back to None. Under Time change Duration to for 1 timepoint. These changes optimize the settings and will be explained in a later section. 9. From the top left menu, go to Video>Acquisition Setup. On the left side of the window, check the change using light paths box and click + until there are as many rows as channels you would like to capture with. Select the channels you would like to capture with 10. At this point you can also name your images and the software will automatically number each item. Type the name in the Title box.

3 Setting up the microscope: 1. Make sure the lenses you will be using are attached to the microscope, magnification and other details are printed on the lens a. For mounted slides, use the air objectives (10X, 20X, 40X) or the oil immersion lens (63X) b. For samples in water, cell culture medium, or PBS, use the water immersion lenses with the beige tips (20X, 40X, 63X) DO NOT use products on lenses that they were not built for. Ex: no oil on any lens except for the oil lens and no waqter for any lens except for the water lenses Water Air Oil If for ANY reason, you are unsure of how to change the lenses or how to use them DO NOT GUESS feel free to ask anyone nearby for help. They are quite expensive Make sure the stage is fully lowered by pressing and holding the down arrow on the left side of the microscope 3. Place your sample on the microscope stage. If your sample is in a culture dish, you will have to place a microscope slide into the microscope slide spot and place your sample on top of the slide 4. If your sample is stained with DAPI, it is best to put your sample into focus using this stain. Turn the filter wheel to 3 for the DAPI filter and pull out the flat black handle so the epifluorescent light can reach the sample. a. You can also use the FITC or RHO filters here but if the signal is weak you will have a hard time finding the correct focus 5. Make sure the metal rod is pushed in, so the image is diverted to the eye pieces. When the rod is pulled out, the image is directed towards the camera. Also make sure the black slider is in the out position so epifluorescent light can pass through 6. Use the stage manipulators and the focus to observe your sample, switching between filters to image 568 or 488 if needed. 2 5

4 Capturing an image: 1. Pull out the metal rod that divers the image, mentioned on the previous page, step 5 and push in the black slider 2. You must check all of the channels you would like to image. Check them by clicking on the red or green circle to view the 568 or 488 channels, respectively. Turn on the laser emission by lifting and pivoting the middle switch on the laser remote control (move from standby to normal). Push in the black slider to make sure there is no epifluorescence coming through. Switch the filter wheel to an empty slot (4 or 5) 3. Gradually increase the laser intensity to just under halfway. 4. If your image is too dim or bright, you can change the exposure time. Be sure to click the save button so the same exposure time will be used when capturing the image. a. You can change the scale (1s or 10s) by clicking on the clock face on the right 8 5. You can also use the live enhancement tool but be aware that this applies changes to all of the channels. You can use this by making sure the black and white circle at the top middle of the window is selected. a. To change brightness, click on the image and drag the cursor left or right 4 b. To change the contrast, click on the image, hold down the control key and drag the cursor up and down. 6. If needed, check the DAPI signal as well. To look at DAPI on the software, click on the blue 6 and set up the microscope to use the epifluorescent light source (see previous page step 5). Adjust exposure, brightness, and contrast accordingly. a. Switch the filter to spot 3, pull out the black slider, and put the laser on standby. 7. When all of the channels look good, set up the microscope to image from the laser first. a. Filter wheel set to 4 or 5, metal rod in the out position, black slider in the in position, laser switched to normal 8. Click the red circle/record button. If you are set up to capture DAPI, the system will pause before imaging the DAPI so you can switch the laser to standby, pull out the black slider, and switch the filter wheel to 3.

5 Creating a Z-stack A Z-stack is a collection of images taken at different Z positions. This technique is typically used to look at cells/specimens that do not lay flat. These images can be exported as a single stacked image or as individual images. 1. Set up the software as you normally would 2. While video preview is selected, go to video>acquisition setup. On the right side of the menu, switch Change focus using to Zeiss focus drive and click OK. 3. Under Zeiss Axiovert/Axioplan on the right hand side of the main screen, click on the button with three arrows (up, down, right). Click on Set zero. a. Clicking set zero sets the reference point for subsequent measurements. If this is not clicked when the microscope focus is within range of your sample, capturing a z-stack will likely break your slide and damage other lenses 4. Change the focus of your image so you are at the topmost frame you wish to image. Click on set top. Do the same for the bottom slice of your image stack and click on set bottom. 5. Record image as usual. a. This process may take a while depending on what your exposure is set to. Capturing Video 1. Set up system for imaging as you normally would. 2. Under Video>Acquisition setup go to the Time tab. 3. Under Timelapse select Use XXX timepoints per second. If you are trying to capture something very fast, 500 timepoints per second is recommended as a good starting place. This can be adjusted as needed 4. Under Duration select Until stop is clicked 5. Make sure your expose is low enough to capture what you would like to. a. If you are looking to capture come kind of repetitive event, such as muscle contraction or action potential propagation, your exposure time must be shorter than the time it takes for the event to happen, likely many times shorter 6. Capture record as usual. 7. When exporting, be sure to export at your desired frames per second a. Item as.wmv is exported at 9 frames per second for real time playback Exporting files The method of export depends on the file you would like to export and the downstream analysis you plan to use. For exporting multiple images, select the images, right click and click export. Save the images on the shared network as view as jpeg (personal preference) or any other file type that works for your analysis. If you cannot find the network, ask Ted to map it out for you. If you want to export individual channels of an image, you can toggle the channels on and off on the right side once an image is selected. Once the channel(s) you wish to export are in view, you can right click and export as usual, giving the file a unique name.