Page 257 - Fundamentals of Light Microscopy and Electronic Imaging

240 VIDEO MICROSCOPY

range of available gray levels from black to white. Given that there is adequate illu-
mination, it is usually desirable to utilize the full range of the camera and make minor
adjustments for brightness and contrast later (if necessary) using an image processor.
If the camera’s signal is not adjusted properly or spans a limited range of amplitudes,
it may not be possible to adjust the gray-level range subsequently using the processor.
Increasing the gain or contrast control amplifies the video signal (multiplies the sig-
nal by a constant) and brings the bright features close to the white or saturated value
on the monitor. Because the signal is amplified by multiplication, the voltage range
corresponding to the black and white end points in the image is increased. For exam-
ple, a signal ranging from 0 to 10 multiplied by 10 becomes 0 to 100, and the voltage
range is increased 10-fold. A sketch showing the effects of gain and offset adjust-
ments on the PMTs of a confocal microscope is shown in Figure 12-12.
• Offset. The offset or black level is a brightness control that is used to set dark fea-
tures in the image close to the black level on the monitor. Offset works differently
from gain. In this adjustment a positive or negative voltage is added to the image
signal to adjust the amplitude of the darkest features in an image to a voltage corre-
sponding to black. Offset changes the amplitude of the entire voltage signal, but
since it is added to the signal, it does not change the voltage difference between
high- and low-voltage amplitudes contained in the original signal. For example, a
signal ranging from 0 to 10 volts modified with an offset of 5 volts becomes 5 to
15 volts, but the difference remains 10 volts as it was originally.
Both gain and offset affect the contrast of the video image. Video contrast C V
is defined as
C A I I /(I I ),
B
B
S
V
V
where A is the amplitude factor for gain, and I is the intensity corresponding to the
V
voltage offset, and I and I are the intensity of the signal and background as
S
B
described in the definition of image contrast in Chapter 2. From the equation it is
clear that both gain and offset determine image contrast. We return to the effect of
these controls later in the chapter.
• Gamma ( ). In the ideal case for an electronic camera, there is a linear relation
between the intensity of incident light on the camera and the strength of the cam-
era’s output signal. This relationship is described by the function,

i/i (I/I ) ,
D
D
where i and I are the output current and corresponding light intensity for a given
signal, i and I are the dark current and its corresponding intensity, and is an
D
D
exponent. Thus, in a plot of the log of the output current of the camera vs. the log
of the light intensity on the camera target, is the slope of the curve. If has a value
of 1, the relationship is linear (Fig. 13-7). Most video cameras have a close to this
value. A setting 1 emphasizes dark features in the image (makes them brighter
and more visible), and has the effect of flattening the image and decreasing contrast
between bright and midgray values. A setting 1 emphasizes bright features and
increases contrast. For the use of scaling in image processing, see Chapter 15.
• Auto-gain control. This feature automatically adjusts the gain to maintain image
brightness. It is very convenient when monitoring fluorescent specimens that are