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EDAS Digital Imaging

USING THE KODAK EDAS 290 DIGITAL IMAGING SYSTEM AND 1D SOFTWARE PACKAGE

EQUIPMENT SETUP
Generally, these steps will not need to be performed for each gel. They should remain constant from day to day. If users have changed settings for a specific purpose, the equipment should be returned to these settings for general use. If you are having problems with your images, checking these settings would be a good place to start.

TRANSILLUMINATOR:
With the POWER switch (Green) set to OFF, the Bulb Intensity switch (Black) should be set to HI.

CAMERA:
Silver rotating switch on the back of the camera must be switched to CONNECT with the 1D program.
Make sure the +3 diopter lens is mounted in front of the camera lens, and the Ethidium Bromide (590nm) filter is in the filter tray.
Aperture should be all the way open in order to let the most light in and keep scan times low.
To set aperture:
1. Rotate silver switch to CAPTURE
2. Press the MENU button on the back of the camera
3. Use the grey cursor button in the center of the rotating silver switch to scroll right until the ADVANCED EXPOSURE MODES menu appears.
4. Scroll down to EXTERNAL FLASH. Press the silver SELECT button at the bottom left corner of the screen.
5. In the new screen, use the cursor button to scroll left or right to change the aperture. F3 is the setting that should be used for quickest scans. Press DONE when the correct aperture is selected. Press DONE a second time to exit the MENU mode and save your settings.
6. Switch the camera back to CONNECT.

GEL LOADING
1. With the power off, lift the door of the gel chamber and gently place your gel in the center of the transilluminator box.
*NOTE* For maximum use of the available pixels, the gel should be placed with its longest sides running left to right and the shortest sides running front to back.
2. Close the sample chamber door.

OPENING THE SOFTWARE
1. On the desktop, select the SHORTCUT TO KODAK 1D 3.5
2. Under the FILE menu, select NEW DIGITAL CAMERA CAPTURE
3. In the SELECT DIGITAL CAMERA window that will appear, drag down DIGITAL CAMERA to select DC290. Port should automatically configure to USB, and connection speed should configure to AUTO. Select OK.

IMAGE CAPTURE
1. Once the sample is loaded, press the IMAGE CAPTURE button located at the top left corner of the screen. A new window will open.
2. Select the sample type and sample size settings appropriate for your gel using the drag down menus.
3. Select your exposure time. 3.5 seconds is a good starting point for a sample of unknown intensity using either Ethidium Bromide or SYBR Gold.
3a. At this point, you can check the positioning of your gel by turning on the transilluminator and selecting the TAKE PREVIEW button. Check for image sizing. For best resolution, the gel should take up most of the screen without being cut off. You can change the orientation (rotation) of your image by selecting one of the four R buttons in the bottom right corner of the screen. Orientation can also be manually adjusted after image capture by selecting ROTATION in the 1D image viewing screen. Be sure to turn off the transilluminator after the image is captured.
*NOTE*
Standard imaging using the "TAKE PICTURE" button seems to result in poor image quality. It is sufficient to check your gel position, and to see if DNA is present etc., but for data analysis, or preparing images for publication, I would suggest using the modified method outlined here. Using the method below will result in the formation of a color TIFF file which is then saved. This TIFF file is then re-opened from within 1D. This method results in much higher resolution and contrast, especially in faint bands.

4A. Turn on the UV transilluminator. Wait for the 4 indicator lights on the front of the UV box to stop flickering and emit a steady blue glow.
4B. Press the EXPORT IMAGE button.

*Note* To minimize DNA degradation due to UV rays, the transilluminator should be kept on as little as possible while the sample is in place. After you have pushed the EXPORT IMAGE button, a status bar will appear saying "Exporting Image". This will remain on much longer than it actually takes to capture the image. The UV box can be turned off long before this status bar disappears. The actual image capture begins almost instantly after pressing the EXPORT image button and will only last as long as the exposure time you selected. You can get a feel for the timing by looking at the LCD screen on the top of the camera itself. The two lines which spin while the camera is idle will become immobile while the picture is being taken.

4c. Once the image capture is complete, a new window will open. You may select a destination folder, name, and image type for your scan. Both JPEG and TIFF file types work well. The TIFF format will result in a slightly higher resolution as it is an uncompressed format, but the difference is minimal.

IMAGE PROCESSING
You now have a raw image which will show up extremely well as a color photo. However, in order to convert it to a printable black and white file, as well as to use the analytic capabilities of the 1D program, a few additional steps are necessary to get the best image quality possible.

1. Under the FILE menu at the top of the screen, select OPEN.
2. Find the location where you saved your image and select it. It should now open as a black and white image on the screen.
3. You can adjust the size of the image for your needs by using the horizontal drag bar located at the bottom left corner of the image screen.
4. Under the SHOW menu, select IMAGE DISPLAY. A new window will pop up. This is the window that allows you to modify the visual characteristics of your image.

The bands seem to be easiest to visualize when the image tonal range is compressed a bit, giving a steeper histogram plot. A good starting point seems to be with the Min (Green Box) set between 20 and 40, Max.(Blue) between 120 and 150, and the Gamma (Red) between 0.7 and 1.0. This is by no means an exact science, and you will just have to tinker with your image settings until you find an acceptable result.

IMAGE EXPORT
If you wish to save your image in a format that can be utilized by other analysis programs or by publication programs such as Powerpoint, Photoshop, or Word, it will need to be converted again out of the 1D program format.

To accomplish this, rather than selecting the SAVE command under the FILE menu, instead select EXPORT DATA, then select IMAGE from the popup menu.

When the SAVE AS dialog box appears, simply select the file type appropriate to your needs in the drag-down menu at the bottom of the window. Select an image name, and location where you want the image to be saved, and click SAVE.
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A portion of AGI's material is based upon work supported by the National Science Foundation under Grant Number 102620.