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Research Technologies Branch

Biological Imaging Section

Protocols: DMIRBE Epifluorescence Microscope

Startup Procedure

  1. Switch on the epifluorescence lamp power supply (on right on shelf above the microscope table) if you are going to image fluorescent probes. This should start almost immediately. This must be switched on before the camera or the computer to prevent serious damage to either.
  2. Switch on microscope. The switch is on the left side near the front. This will also turn on power for the shutter filter wheel control.
  3. Turn on the joystick stage. This is the flat white control box on the shelf behind and to the left of the microscope. The power switch is on the back of the box, near where the power cord connects.
  4. Switch on the computer with the white button on the front. This is the white box beneath the bench. Switch on the monitor and allow the computer to complete its boot sequence.
  5. Log onto the network with your usual network password and name. Make sure the domain is NIAID.
  6. Switch on the color filter wheel control if you are going to collect color images. This is the small white CRI box atop the old camera control box. There is a small rectangular button to press in to turn it on.
  7. Switch on the camera with the switch on the top of the camera. A red LED will start flashing. This will turn to green when the camera temperature stabilizes.
  8. Click start --> programs --> Image Pro Plus 4.5 --> Image Pro Plus to load the image acquisition package.
  9. Click on the color camera icon in the toolbar to open the camera control dialog box.
  10. In the camera dialog box, select 8 bit B & W if you are collecting a black and white, or single color, fluorescent image. Collecting fluorescent images without the color LCD greatly reduces exposure (integration) times. Make sure the LCD filter slider is in the out position.
  11. To view samples, open the fluorescence shutter with the right button on the control box beside the microscope. (The left two buttons are used to select the excitation wavelength.) Make sure the slider near the oculars is pressed in all the way.
  12. If you are not familiar with the microscope's operation, consult the instructions that follow for the DMIRBE microscope. Note: the 20xLWD and 40xLWD (dry) lenses have compensation collars. These must be adjusted to get sharply focused images.

Image Collection

  1. Make sure the fluorescence or white light source is open to the specimen. Pull the slider beside the oculars to the left, to the full open position. This directs light to the CCD camera.
  2. In the camera control dialog box, click preview for focus. Focus by moving the focus knob on the microscope until the image is sharp. When the object is in sharp focus, click stop preview.
  3. Click snap to collect the final image. As soon as the image collection is completed, close the fluorescence shutter to prevent bleaching of the sample.
  4. You will have to enter an exposure time for preview and for acquire. Make sure that the autoexposure is turned off when doing 8-bit greyscale collection. This control is found under the acquire tab, autoexposure options button; make sure the box autoexpose is not checked. The filter should be set to none.
  5. There is a box under the "preview" and "acquire" tabs marked "lock exposure times." This sets the preview and acquire times the same. You can chose to use or not use this option, depending on your preference.
  6. To set exposure time, click start preview and check the image. If it is too bright, click stop preview, enter a reduced exposure time, and click start preview to check. You must stop the preview before entering a new exposure time. Repeat this process until a satisfactory time is selected.

Color Image Collection

Use this for very bright fluorescent images or brightfield histology samples. The software will collect separate exposures for red, green, and blue, and combine them to a single color image.

  1. In the camera control dialog box, click 24 bit color.
  2. Slide the color LCD filter slider into the light path. (It is beneath the camera).

Colorize and Merge Files

  1. Open all of the files that need to be colorized.
  2. Select colorize grey scale images. This is the ninth button from the left, and is symbolized by a series of colored bars. In the division box, type 256, then select . Next, under color spread, select the desired color (i.e., green, red etc.), then click apply.
  3. The final step is to merge the single color images. Select process --> color channel, then select the merge tab. Select the colors involved by "checking" the channel boxes. Then select the file for each channel. Then click OK. This will give a new file with all the specified colors. Name and save this file.

Save files on drive D: to create a user area. Transfer the files when you are done.

If collecting DIC images, be sure to pull the polarizer out before collecting fluorescent images.

Note: Be sure to switch everything off—including the camera, stage, CRI color filter, and such—when you are done.

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Last Updated April 05, 2007