Page 273 - Fundamentals of Light Microscopy and Electronic Imaging

256 VIDEO MICROSCOPY

3. A raw image of the cells will appear on both TV screens. One screen dis-
plays the raw, unprocessed image while the other displays a processed
image from the image processor. If the screen of the raw image monitor is
black, the light is too dim or the connections and/or circuits are not set
correctly; if the screen is white, the camera is oversaturated. Reduce the
light intensity to the camera until a normal image appears on the screen.
Do no oversaturate the camera! Most camera controllers have a light that
indicates when illumination intensity for the camera is too dim, in the
optimal working range, or saturating.
4. Special UV and IR cut filters should be placed in the illumination pathway
to protect the cells from photodamage. A green interference bandpass fil-
ter centered at 546 nm should also be used to further protect the cells and
improve image contrast. Brightness and contrast of the raw image are
adjusted using the offset and gain controls on the camera controller before
further adjustments are made with the digital image processor.
• Adjust the digital image processor for frame averaging, background subtrac-
tion, and digital contrast adjustment.
1. Position the specimen and use the gain/offset dials on the camera con-
troller to optimize the raw image. Adjust the offset first followed by the
gain.
2. Click/select the background subtraction function on the image processor.
3. Select the frame number 4, 8, or 16 for frame averaging. This setting
selects the number of video frames that will be used for frame averaging.
Image quality improves dramatically because the S/N ratio increases as
the square root of the number of frames averaged. For dim, noisy images,
frame averaging can make a tremendous improvement, although averag-
ing reduces the temporal resolution and may not be suitable for rapidly
moving objects. (Another way to see the same effect is to select the oper-
ation called averaging. Simply select the number of frames and hit the
start button to see the effect.)
4. Find a clear area of background next to and in the same focal plane as the
cell. If there are no clear areas, defocus the specimen by a small amount.
Click “Background” to store the background image in the frame buffer of
the processor. Then press “Start” to initiate subtraction. The screen will
turn blank and medium gray. Reposition the cell and refocus if necessary.
You are now performing a simultaneous operation of frame averaging and
background subtraction. This operation increases the S/N ratio and
smoothness of the image and removes images of dust and optical blem-
ishes in the microscope-camera system.
5. After the camera’s offset and gain controls have been optimized, you may
make further improvements in brightness/contrast in the processed image
using the digital contrast adjustments on the processor. Be sure the image
is being processed for frame averaging and background subtraction. Now
click on the “Enhance” button on the processor to make further adjust-