Keck Microscopy Facility

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Collecting Images

1) Log onto the computer
Name: Keck
Pass word: User

2) Turn on the HG lamp if you’re imaging fluorescence.
Turn on POWER
Press and hold IGNITION button until LAMP READY light comes on

3) Turn on the camera.

4) Turn on the microscope.

5) Explore your specimen using the appropriate fluorescent filter cubes. When you find something you want to image, open QCapture.

6) Fully extend the light path selector to send light to the camera.

7) Click on the green arrow (Start/Stop Preview) on the Qcapture toolbar to start the preview window.

8) Click on the Magnifying glass with the negative sign to reduce the size of the preview window. Note: You can also use the scroll bars to find a region of interest within the larger preview window.

9) The Camera Settings window will open with the default settings for Format (Monochrome), Preview Depth (8 bit) and Capture Depth (8 bit). These settings are what you should use for fluorescence imaging. Leave the Binning set to 1 for both dimensions unless you have a weak signal and are willing to sacrifice resolution for higher signal/noise.

10) Click the Exposure tab and click Auto Exposure to get an initial exposure.

11) Refine the initial exposure:

Drag the Exposure slider to shorter or longer exposure times to get the right-hand portion of the graphic readout to near the maximum brightness level without clipping off data (saturating the camera).

Drag the Offset slider to shorter or longer settings to get the left-hand portion of the graphic readout to near the minimum brightness level without clipping off data.

Note that the scale for the sliders is not linear! Clicking on the scale near the slider may be an easier way to go when you just want to move it a small amount.

12) Focus and frame the specimen using the microscope focus and stage controls.
Note: Leave the Gain at 1 unless you are getting exposure times that are too long to be able to focus the camera. Increasing the Gain and decreasing the Exposure will result in noisier images.

13) When you have the correct exposure settings, record them if you need to make a series of identical exposures, then click on the camera icon (Snap) on the Qcapture toolbar. This will collect the image into QCapture.
Note: The Preview window will remain open and may obscure your
images.

14) Change the filtercube and repeat steps 10 – 13 for each fluorophore in your specimen.

15) Periodically save you images. If QCapture hangs up before you save animage, it will be lost.
Note: Do not leave images on the image collection computer! They may be deleted at anytime, without warning!

Saving Images

1) Click on the disk icon (Save Image) on the QCapture toolbar or select File ? Save As.

2) Select or create a directory to save the image in, name the image andleave Save as type: set to TIFF.

3) Select “Save only the raw data” and “Least-Significant-Bit aligned”
Note: “Most-Significant-Bit aligned will save the image as 16-bit.
“Save image as displayed on the screen” will change your image data if you have manipulated the image prior to saving the image.

4) Hit Save.


 

Contact Greg Martin at gvm@u.washington.edu for additional information. Suggestions and comments are always welcome!

© 2006 Keck Imaging Center, University of Washington