Page 268 - Fundamentals of Light Microscopy and Electronic Imaging

VCRS 251

A better solution for low-light imaging is the insertion of a high-resolution, high-
gain image intensifier between the microscope and the camera. Image intensifiers
increase the intensity of dim fluorescent specimens and are named Gen I, II, III, or IV
according to their design and performance specifications. Gen II and Gen III intensifiers
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give an electronic amplification (gain) of 10 –10 and generate up to 30- to 50-fold
increases in the light intensity delivered to a camera that is coupled to the intensifier
with an intervening lens. The target at the front end of the intensifier tube receives inci-
dent photons and emits electrons, which are accelerated onto and through a microchan-
nel plate to further amplify the number of electrons. The electrons excite a phosphor
screen at the exit end of the microchannel plate, generating the intensified image. The
screen’s image is then projected onto the video camera with a relay lens. Alternatively,
the intensifier’s phosphor screen can be directly coupled by optically transparent cement
to the detector (such as a CCD chip) by a large-diameter fiber optic bundle. Details on
the design and performance of intensifiers are presented in Video Microscopy by Inoué
and Spring (1997). There are several points worth noting about intensifier performance:

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• Although the electronic gain of a Gen II intensifier is very large (10 –10 ), the increase
in light intensity delivered to a lens-coupled camera is usually considerably less, typi-
cally 10- to 30-fold. This decrease occurs because the practical working range of the
electronic gain is considerably less than the potential maximum and because of the low
efficiency of signal transfer from the intensifier phosphor screen to the relay lens and
camera detector. Since intensifiers are usually operated at 10–50% of maximum volt-
age to preserve their life, the observed increase in light amplification by a gen II inten-
sifier is 10- to 30-fold under typical operating conditions. Despite the losses, a 10- to
30-fold increase in light intensity can be extremely valuable.
• Image fidelity can be altered by the microchannel plate, which, due to the hexago-
nal packing of its capillaries, contributes a hexagonal chicken-wire pattern to bright
images. Newer Gen III intensifier designs minimize this problem by using smaller
capillaries.
• Increasing the intensifier gain changes the range of sensitivity of the intensifier, but
because the intensities of all image points are amplified by a constant factor (gain),
the intrascenic dynamic range (the range within a single image view) remains the
same. However, changing the gain increases the interscenic dynamic range consid-
erably (the total dynamic range represented by images obtained under a range of
lighting conditions).

Despite some limitations, image intensifiers allow video detection and display of
fluorescent objects that are at or below the limit of vision while looking in the micro-
scope. Certain Gen III intensifiers are capable of showing individual GFP or fluorescein
molecules at the video rate of 30 frames/s! Intensifier-coupled cameras are employed
for examining single-molecule dynamics using total internal reflection fluorescence
(TIRF) microscopy. For further orientation, see Spring (1990).

VCRS

Because of limitations in reading and writing to magnetic tape medium, images
recorded on a video cassette recorder or VCR have reduced spatial resolution compared
to that inherent to the original video signal. The signal response and bandwidth of a