Page 49 - Fundamentals of Light Microscopy and Electronic Imaging

32 ILLUMINATORS, FILTERS, AND THE ISOLATION OF SPECIFIC WAVELENGTHS

cial tungsten film and photographic filters are available to correct for the red-rich spec-
tral output of these lamps.
Ion arc lamps are 10–100 times brighter than incandescent lamps and can provide
brilliant monochromatic illumination when combined with an appropriate filter, but the
increase in brightness comes with some inconveniences in mechanical alignment,
shorter lifetime, and higher cost. Two types are commonly used: 75 W xenon and 100 W
mercury arc lamps. Both lamps produce continuous spectra across the entire visible
range from 400–750 nm and extending into the ultraviolet and infrared. In fact, only
about a fifth of the output of these arc lamps is in the visible portion of the spectrum, the
remainder being in the ultraviolet and infrared, so special blocking filters must be used
when examining living cells, which are sensitive to UV and IR radiation.
Arc lamps tend to flicker due to subtle variations in power. This can be annoying,
especially during time lapse recording, but stabilized power supplies are now available
that minimize this problem. It is also common practice to avoid turning the lamp on and
off frequently, as this poses a risk to nearby electronic equipment and shortens the life
of the bulb. If there is a 20–30 min pause in the use of the microscope, it is better to
leave the lamp on than to turn it off and reignite it. There are, however, new lamp
designs that use a variable transformer to control light intensity. As the bulb is turned
down, a heating mechanism keeps the lamp hot so that brightness increases immediately
when the power is turned up again. The lifetime of mercury and xenon lamps is gener-
ally rated at 200 hours; however, the UV output of a mercury lamp weakens (sometimes
considerably) with prolonged use, since metal vapors from the electrodes become
deposited on the glass envelope. In addition, the arc becomes unstable and begins to
flicker. Although arc lamps are expensive, the actual cost works out to be about 50 cents
per hour, so it is advisable to replace them after their nominal lifetime has expired even
if they are still working.
The mercury arc lamp is distinct in emitting several prominent emission lines, some
of which are up to 100 times brighter than the continuous background: 254 (far UV),
366 (near UV), 405 (violet), 435 (deep blue), 546 (yellow-green), 578, 579 (yellow dou-
blet band), plus several lines in the IR. The 546 nm green line of the mercury arc lamp
is a universal reference for calibrating wavelengths in a number of optical devices and is
a favorite among biologists for examining living cells. UV emission accounts for about
half of the output of the mercury lamp, so care must be taken to protect the eyes and liv-
ing cells that are illuminated by it. When changing and aligning a new lamp, avoid star-
ing at the unattenuated beam; when examining live cells, use a green bandpass filter plus
a UV-blocking filter such as a Schott GG420 glass filter. Since mercury lamps also emit
in the IR, heat-cut filters are desirable to block these wavelengths as well.
The spectrum of the xenon arc lamp is largely continuous and lacks prominent
emission lines. Its advantage is bright, uniform output across the entire range of visual
wavelengths. At blue-green and red wavelengths it is significantly brighter than a 100 W
mercury lamp, making it advantageous for certain applications in fluorescence
microscopy. Since about half of the light emitted from a xenon lamp is in the IR, special
IR-blocking filters, such as a Schott BG38 or BG39 glass filter and/or an IR-reflecting
mirror, are used to attenuate and block these wavelengths and protect cells from excess
heat. The detectors of electronic cameras, particularly those of CCD cameras, are also
particularly sensitive to infrared light, which can fog the image. Although the intensity
of a 75 W xenon lamp is high, the distance between the lamp electrodes is small—only
0.75 mm—which can make it difficult to obtain an even distribution of the light across
the front aperture of the condenser and therefore across the specimen in the object plane.