Page 209 - Fundamentals of Light Microscopy and Electronic Imaging

192 FLUORESCENCE MICROSCOPY

One of the most important adjustments in fluorescence microscopy is the alignment
of the illuminator. Since the amplitude of fluorescence signals depends directly on the
amount of excitation, uneven illumination of the object by a misaligned lamp will result
in bright and dark regions in the fluorescence image. This is especially detrimental for
quantitative work. Lamp alignment is discussed in Chapter 3 and demands attention to
two points: (1) centration of the image of the arc and its reflection on the optic axis, and
(2) spreading of the illumination beam with the lamp collector lens to fill the back aper-
ture of the objective evenly and homogeneously.

Filters

Filters used to isolate bands of wavelengths in fluorescence microscopy include colored
glass filters, thin film interference filters, or a combination of filter types. The filters
include long-pass or short-pass edge filters or narrow or broad bandpass filters. The
design and action of filters are discussed in Chapter 2; the optical performance of repre-
sentative filters used for fluorescence microscopy is shown in Figure 11-8 and in Color
Plate 11-1. It is essential to understand the properties and value of these indispensable
components of the fluorescence microscope. You will need to know how to “read” the
absorption and emission spectra of a dye, and the transmission profiles of the filters.
This knowledge is required to select the best among several possible filter sets for exam-
ining the fluorescence of a certain fluorochrome. You will also need to interpret and
explain differences among fluorescence microscope images obtained using different fil-
ter sets. In addition, you may need to create new filter sets using components of filter
sets already in hand. This will greatly extend the capacity of a limited collection of fil-
ters. As you install and remount filters, remember that interference filters are usually
mounted in a specific orientation with respect to the illuminator and the specimen in
order to obtain optimal performance (see Chapter 2). The transmission profiles of filter
sets for fluorescence microscopy can be examined at the Web sites of Chroma, Inc., at
www.chroma.com, and of Omega Optical, Inc., at www.omegafilters.com.

The Dichroic Mirror

The dichroic mirror or beam splitter is a special long-pass filter coated with multiple lay-
ers of dielectric materials similar to those contained in thin film interference filters, but
specially designed for reflection and transmission at certain boundary wavelengths. The
mirror is mounted at a 45° angle with respect to the optic axis within a filter cube and
faces the light source. At this angle, the dichroic mirror reflects short excitation wave-
lengths at a 90° angle along the optic axis to the specimen, but transmits long fluores-
cence wavelengths that are collected by the objective and directed to the image plane.
The transition from near total reflection to maximal transmission can be remarkably
sharp, occurring over 20–30 nm, allowing the mirrors to act as precise discriminators of
excitation and fluorescence wavelengths (Fig. 11-8). (For a description of the design and
performance of interference filters, see Chapter 2.) The specifications for a dichroic mir-
ror assume a 45° angle between the axis of the incident beam and the plane surface of the
mirror. Dichroic mirrors should be handled with extreme care, because the exposed
dielectric layers can be scratched and damaged during handling and cleaning. The clean-
ing procedure is the same as that used for cleaning other optical surfaces (Chapter 2).