Page 267 - Fundamentals of Light Microscopy and Electronic Imaging

250 VIDEO MICROSCOPY

Spatial filtering: Enhances high or low spatial frequency information. Masks com-
posed of various pixel matrices are used for edge enhancement, shadow enhance-
ment, sharpening, or smoothing of the image.
Image trace: Traces the trajectories of moving objects.
Maximum density trace: Traces the pathways of moving objects that are brighter
than the background.
Minimum density trace: Traces the pathways of moving objects that are darker than
the background.

The image display commands of the processor are also versatile and include con-
trast adjustment with image histogram display, with adjustments for setting the white
and black end points; adjustment for differential adjustment of bright and dark fea-
tures; real-time edge sharpening; split-screen display; pseudocolor; superimposition of
a real-time image over an image stored in memory; real-time zooming; superimposed
display of scale bar, typed comment, and timer/clock.
Image processors also provide image analysis features for measuring lengths of
traced lines, areas, particle counts, interpoint distances, and velocities. Intensity analy-
sis commands allow you to measure intensities of points and average intensities within
areas; you can also obtain intensity profiles along a line through an image, an image his-
togram of a designated region of interest, and a three-dimensional profile of intensity
levels within a defined region of interest.

IMAGE INTENSIFIERS

Although the temporal resolution of video cameras is excellent, their sensitivity to light
is limited. This is because their quantum efficiency is only moderate (20–30%), the
exposure time is short (fixed at 33 ms), and because the camera electronic noise asso-
ciated with rapid read rates is high. An image processor placed in-line between the cam-
era and TV monitor can accumulate or average the signals from hundreds of frames for
display on the TV, but electronic noise limits its ability to detect faint signals. Even with
frame averaging and high gain, the detection limit (as judged from a noisy, barely
acceptable image) corresponds roughly to an object of moderate fluorescence intensity
as seen through the eyepieces of a fluorescence microscope. Faint fluorescent objects
cannot be detected at all. The solution is to use a low-light-level camera or a camera cou-
pled to an image intensifier.
Silicon intensifier target (SIT) and intensifier silicon-intensifier target (ISIT) cam-
eras have 10- to 100-fold higher sensitivity to light than vidicon tube and video CCD
cameras, and have been employed for low-light fluorescence microscopy for many
years. SIT cameras contain a low-light-level vidicon tube that is modified such that pho-
toelectrons generated and emitted at the photocathode at the front end of the tube are
focused and accelerated onto a silicon diode target that responds more efficiently to the
incident electrons than does the standard vidicon tube. ISIT cameras, which can detect
even dimmer objects, contain a Gen I–type intensifier in front of the SIT tube. These
designs give improved sensitivity and greater dynamic range at low light levels. How-
ever, these cameras exhibit a lag in response time that results in the appearance of comet
tails for rapidly moving objects, a falloff in resolution from the center to the periphery
of the image, gain instability, and significant geometric distortion and shading, which
make them less desirable for quantitative applications.