Using the Confocal function Nikon Elements software 1. Turn on: a. Computer (login and password on monitor) b. Microscope c. Light sources (brightfield/fluorescence, as needed) d. APC Box e. Main laser power supply f. Lasers as needed g. C2 controller box – wait for “ready” light before proceeding h. Elements software - From the camera options at startup, select “Nikon Confocal” 2. Find Sample using eyepieces with brightfield or fluorescence 3. Switch to confocal: a. Turn safety switch clockwise: Bino confocal b. Turn off brightfield c. Put blank filter block (position 6) in place d. Remove analyzer e. Set light path to scan head (left) 4. Set up Imaging Parameters: a. On the C2plus Compact GUI control panel, check the boxes for the lasers you are using b. Next to the button for each laser, set the power to 5-10% c. Set the gain (HV) at about halfway for each laser d. Next to “pinhole” click the 1.2AU button for optimal pinhole size e. Click “Find” f. Click on “split components view” button ( ) on top menu bar to see channels separately g. Click the saturation indicator button ( ) on top menu bar to see saturated pixels Blue channel = yellow Green channel = pink Red channel = pale blue h. Adjust the gain (HV) using mouse wheel for each channel until just a few pixels are saturated. Stop “find” i. If merged image is not visible, click the “overlay” button to the right of the “split components view” button 5. Start Imaging: a. On the C2plus Compact GUI control panel, click “Scan” b. Adjust focus to make sure you are at the brightest focal plane of your specimen, and adjust gain so as not to oversaturate c. STOP live scanning when you have finished adjusting gain. 6. Select an area of interest: a. Underneath the preview on the C2plus Scan Area window, the “Nyquist XY button will show you how far you can zoom in while maintaining acceptable resolution. b. Adjust the green square as needed. You can make it bigger, but you should not go any smaller. c. Right click in the preview to confirm the new scan area d. To unzoom, click the reset button 7. Improve Image quality: a. On the C2plus Compact GUI control panel, click on the pulldown arrow on the averaging button b. Choose an average number, eg. 4 c. Click “Capture” d. If the image is still noisy, try increasing the average number. e. To see the image without the saturation indicator, unclick the saturation indicator button ( ) f. To see the merged image only, uncheck the “split components view” button ( ) g. If you want a higher resolution image, change the image size from 512 to 1024 or 2048 (these sizes are not recommended for live scanning as it is too slow) 8. Save the image: a. Go to File>Save as b. Find your folder in the Microscopy folder on the server (shortcut on desktop). NOTE that this computer is frozen. Any files saved on this computer will be deleted upon shutdown c. Make a folder for your session, named by the date in this way: yymmdd d. Save the image as a JPEG 2000 file. Always save a copy in this format. You can save another copy as a tiff file to look at on another computer. Special Functions: capturing series. 1. Capturing a z-series: a. Make sure the scan mode is set to “Normal”, not averaging b. Hit “scan” and focus to the median plane of your specimen or of the part of your specimen that you are interested in. c. On the “ND Acquisition” control panel, uncheck any functions you are not doing (eg. Time) and check Z-series tab d. Focus to the bottom of your specimen (towards coverslip) e. Click “bottom” f. Focus to the top of your specimen g. Click “top” h. STOP SCAN i. Check the number of steps. If it is an insanely high number, you can reduce it by changing the top/bottom points or by increasing the step size (be careful not to make it too big. About half the objective’s depth of field is usually the biggest you can get away with – see sheet on wall for depths of field) j. Choose your averaging mode k. Hit “run now” on ND acquisition control panel. Wait for z-series to complete. 2. Viewing a z-series: a. The buttons shown above can give you different views of your series. The first three will split the channels. The next four give you merged, orthogonal, 3D and individual slices views, respectively. The next two perform maximum and minimum projections (you will almost always want maximum). The last two perform “extended depth of focus”, which is another way to look at a 3D rendering of your z-series b. You can save both the original z-series and the 3D rendering. The 3D rendering can always be recreated from the z-series, so always save the series in ND2 format. The 3D rendering can be saved as a JPG or TIF if you want to open it elsewhere. 3. Capturing a time series: a. On the “ND Acquisition” control panel, uncheck any functions you are not doing (eg. Z series) and check Time tab b. Set the time interval (how often it will take a picture and the total duration (eg. Image every 30 seconds for 20 mins). c. Choose your averaging mode d. Hit “run now” on ND acquisition control panel. Wait for the time series to complete. 4. Some tips for time series: a. If you are using “average”, the “frame time” will be longer. The “Frame time that appears under the averaging setup shows the time for one frame. If you average X4, expect the frame time to actually be four times the frame time shown. Your fixed delay interval must be longer than the total frame time. b. To speed up the frame time, you can try: i. Reducing the average number ii. Moving the top and bottom of the bounding box (if zooming) inwards so that the frame is a rectangle wider than tall iii. Reducing the pixel dwell (if it is not already set to fastest) 5. Viewing a time series a. Click the Play button at the bottom of the image window. The “-“ and “+” next to it will change the playback speed. b. Time series can be saved as .avi files, but make sure you FIRST save it as an ND2 file! 6. Shutting down a. Save images b. Exit software c. Answer no to all dialog boxes unless you know you want to change your profile d. Turn off green laser (key ONLY) e. Turn off C1 controller box f. Turn off light sources g. Return microscope to brightfield settings: i. Confocal bino ii. Light path to eyes iii. Brightfield light on h. Clean oil off objectives, set microscope to low power objective i. Turn off microscope and cover j. Check the booking schedule to see if anyone is booked on after you. If you are finishing early, adjust your booking. k. Shut down computer (if no one is booked after you) l. When fan on lasers stops, turn of green laser at switch, and other lasers m. Turn off main laser power supply n. Turn off APC box o. Clean up any lens paper, kimwipes, slides etc that you used.
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