Page 211 - Fundamentals of Light Microscopy and Electronic Imaging

194 FLUORESCENCE MICROSCOPY

Transmission profiles for dichroic mirrors usually show multiple broad peaks and
troughs that correspond to bands of wavelengths that experience high transmittance/low
reflectance (at peaks) and low transmittance/high reflectance (at troughs) (Fig. 11-8).
Filter sets are designed so that the band of excitation wavelengths (high-percent trans-
mission) from the exciter precisely matches a trough in the dichroic (low-percent trans-
mission) so that these wavelengths are reflected to the specimen. Longer fluorescent
wavelengths emitted by the specimen must also match the peak to the right of the trough
so that they are transmitted to the emission filter and detector. It is important that the
transmission, reflectance, and emission characteristics of the exciter and dichroic be
closely matched, and that they be appropriate for the absorption and emission maxima
of the dye; otherwise, excitation wavelengths can pass through the dichroic and fog the
image, or fluorescent wavelengths can be reflected at the dichroic, reducing image
brightness. Even when filters and fluorochromes are appropriately matched, perfor-
mance is usually compromised somewhat if the transmission profiles of the exciter and
dichroic overlap. When this happens, some excitation light passes through the dichroic,
reflects off the walls of the filter cube, and can be partially transmitted by the emission
filter, because the angle of incidence with that filter is oblique. Transmission of
unwanted wavelengths through a filter set is called bleed-through, and the amount of
bleed-through for typical filter sets is generally about 10%. Microscope manufacturers
continue to improve fluorescence optical designs to give higher contrast images.
It is important to recognize that in addition to reflecting the excitation band of
wavelengths, a dichroic mirror usually reflects bands of wavelengths shorter than the
excitation band. Therefore, you cannot always depend on the dichroic filter to block
transmission of unwanted short wavelengths, which to a greater or lesser extent always
leak through the exciter filter. It is usually wise to insert an additional UV-blocking fil-
ter into the beam when examining live cells by fluorescence microscopy.
Advances in thin film coating technology allow for creation of multiple transmis-
sion peaks and alternating reflection troughs in a single interference filter or dichroic
mirror. When matched appropriately, two filters and a dichroic mirror can be combined
to create a multiple fluorescence filter set that allows simultaneous excitation and fluo-
rescence transmission of multiple fluorochromes (Fig. 11-9). Multiple-wavelength fil-
ters and dichroic mirrors are now commonly employed in research-grade microscopes
and confocal fluorescence microscope systems (see Chapter 12). Double, triple, and
even quadruple fluorescence filter sets are available for fluorescence microscopy,
although these sets are expensive and suffer somewhat from bleed-through—that is, the
transmission of fluorescence from one dye through bandwidths intended for other dyes.
The clearest multifluorochrome images are obtained by taking separate gray-scale pic-
tures with filter sets optimized for each dye and then combining the images into a single
composite color image, either electronically with a computer or in the darkroom.

OBJECTIVE LENSES AND SPATIAL RESOLUTION
IN FLUORESCENCE MICROSCOPY

Proper selection of an objective lens is important, especially for imaging dim fluores-
cent specimens. High-NA, oil immersion plan-fluorite lenses and planapochromatic
objective lenses are ideal, because at NA 1.3 or 1.4 their light-gathering ability is
especially high. These lenses give excellent color correction, so different fluorescent
wavelengths are brought to the same focus in the focal plane. They are also transparent
to UV light—a requirement for examining UV-excitable dyes such as DAPI, Hoechst,