Page 208 - Fundamentals of Light Microscopy and Electronic Imaging

ARRANGEMENT OF FILTERS AND THE EPI-ILLUMINATOR 191

cube that is positioned along the optic axis. The first component, the excitation filter (or
exciter) selectively transmits a band of short wavelengths (relative to the fluorescence
wavelengths) for exciting a specific fluorochrome in the specimen. The second compo-
nent, the dichroic mirror, reflects the short wavelength excitation light toward the objec-
tive lens and specimen, while transmitting returning long-wave fluorescent light toward
the detector. The dichroic mirror also directs any excitation wavelengths reflected by the
specimen back toward the exciter filter and illuminator. The third component is the
emission or barrier filter, which transmits the band of fluorescence wavelengths while
blocking any residual short excitation wavelengths. The fluorescent wavelengths then
form an image on the eye or camera.

Lamps for Fluorescence Excitation

The excitation source can be a band of wavelengths isolated from a mercury or xenon
arc illuminator with an interference filter, or it can be monochromatic light from a laser,
but illuminators must produce photons of the proper wavelength and at a sufficiently
high intensity to be efficient. To obtain a particular wavelength of the desired intensity,
it may be necessary to use a certain illuminator—for example, a mercury arc excites
DAPI much more effectively than a xenon arc. Therefore, it is important that the micro-
scopist match the illuminator to the fluorescent dye in question.
For fluorescence microscopy, 100 W mercury and 75 W xenon arc lamps are com-
monly used. Both lamps give bright, continuous emission over the visible spectrum
(400–700 nm), but mercury is distinct in containing sharply defined emission lines at
366 (UV), 405, 436, 546, and 578 nm. It is important to reexamine the spectra and char-
acteristics of arc lamps, which are presented in Chapter 3. At 546 nm, mercury and
xenon lamps are 10–100 brighter than a 100 W quartz halogen lamp, which is typi-
cally used for bright-field microscopy, but is too weak to excite most fluorochromes
adequately. The bright emission lines of the mercury arc are useful for exciting certain
fluorochromes such as DAPI and Hoechst dye 33258, lucifer yellow, rhodamine, Cy3,
and Texas red, but at other spectral regions xenon is brighter and may be more useful
(fluorescein, Alexa 488, Fluo3, allophycocyanine, Cy5). Refer to Table 11-1 and Figure
11-5 on fluorescent dyes and the spectra of xenon and mercury light sources in Chapter
3 to confirm this for yourself and to find other well-matched dye-illuminator partners.

The Epi-Illuminator

The epi-illuminator consists of the lamp and its collector lens plus a connector tube fit-
ted with a field stop diaphragm, a relay lens, and slots for additional filters. It is attached
to the lamp at one end and is mounted at its other end to the microscope in the vicinity
of the fluorescence filter cube. The lamp housing should contain a focusable collector
lens to fill the back aperture of the objective as required for Koehler illumination. On
most research microscopes, the epi-illuminator is a built-in component of the micro-
scope body. Apart from proper adjustment and focus of the lamp, little other manipula-
tion of the illumination pathway is required. Check that the field stop diaphragm is
centered and is opened to provide optimal framing of the specimen. Epi-illumination
is much more efficient than transmitted mode (or diascopic) illumination because there
is much less background in the fluorescence image.