Page 46 - Fundamentals of Light Microscopy and Electronic Imaging

CHAPTER
3

ILLUMINATORS, FILTERS,

AND THE ISOLATION

OF SPECIFIC WAVELENGTHS

OVERVIEW

To obtain optimal imaging performance in the light microscope, the specimen must be
properly illuminated. This requires proper selection of wavelength and intensity and
correct alignment and focus of the lamp. The first objective is met by matching the par-
ticular application to the proper combination of illuminator and filters. Since research
microscopes may be equipped with a variety of lamps, including quartz halogen lamps
and other tungsten filament lamps, mercury, xenon, and metal halide arc lamps, we dis-
cuss the energy and spectral output of various illuminators. Filters that adjust light inten-
sity and provide wavelengths of a particular color are also discussed. For example, if the
microscope is equipped with a constant wattage power supply, the intensity must be
controlled using neutral density filters; similarly, colored glass filters and interference
filters are used to isolate specific color bandwidths. It is the combination of illuminator
and filters that determines the quality of light directed to the condenser for illuminating
the specimen. While all forms of light microscopy require selecting illuminators and fil-
ters, knowledge of their action and function becomes especially important in fluores-
cence and confocal fluorescence microscopy. We close the chapter by discussing how
illuminators and filters must be carefully considered when examining living cells.

ILLUMINATORS AND THEIR SPECTRA

Successful imaging requires delivery to the condenser of a focused beam of light that is
bright, evenly distributed, constant in amplitude, and versatile with respect to the range
of wavelengths, convenience, and cost. Alignment and focus of the illuminator are
therefore essential and are the first steps in adjusting the illumination pathway in the
microscope. A number of incandescent filament lamps and arc lamps are available to
meet the needs of various applications. The spectra of the principal lamps used in
microscopy are shown in Figures 3-1 and 3-2 and are summarized here.
Incandescent lamps with tungsten wire filaments and inert argon gas are frequently
used for bright field and phase contrast optics and are bright enough for certain applica-
tions requiring polarized light. Tungsten and quartz halogen lamps are convenient and 29