The Principles of Clinical Cytogenetics - Extra Materials - Springer

Fundamentals of Microscopy 89
FLUORESCENCE MICROSCOPY
Microscopes used for brightfield microscopy can also be equipped for fluorescence microscopy. The
additional components for fluorescence microscopy include an epifluorescence lamp housing, a horizontal
attachment for the fluorescent light path, fluorescence filters, and fluorescence objective lenses.
Essentials of the Fluorescence Microscope
Epifluorescence Lamp Housing and Microscope Attachment
The housing for the epifluorescent light source is mounted on the rear of many microscopes and is
located just above the housing for the brightfield light source. The fluorescence housing is mounted
to an epifluorescent microscope attachment, which is used to direct fluorescent light into the microscope
and to house the fluorescence filters. Most fluorescent lamp housings include bulb alignment
controls and an adjustable collector lens to control the dispersion of the light across the microscope
field of view. The epifluorescent microscope attachment also includes a light shutter to block the
passage of light into the microscope, a field diaphragm to control the area of illumination, and a
means of inserting infrared or neutral density filters.
Filters
Infrared (IR) filters can be used with electronic cameras to block IR light emitted by the lamp and
keep it from from reaching the camera and producing a high background. IR filters can also reduce
the overall intensity of the fluorescent light and should therefore be used on an “as-needed” basis. In
addition, IR filters should not be used with fluorescent dyes that rely on infared or near-infared
wavelengths for excitation.
Neutral density (ND) filters allow the intensity of the fluorescent light of all wavelengths to be
reduced (attenuated) by a fixed amount. Neutral density filters come in several attenuations and are
labeled to indicate the degree of reduction. Neutral density filters are often used with fluorescent
preparations that have unusually bright fluorescence intensity or that produce a great deal of fluorescent
flare. Neutral density filters can dramatically reduce the intensity of a limited fluorescent light
supply and should be used only when necessary.
Fluorescence Filters and the Fluorescence Filter Housing
The basic principal of fluorescence microscopy involves the excitation of a fluorochrome (fluorescent
stain) with one wavelength of light, causing the emission of a second wavelength of light. The
wavelength used for excitation will vary for each fluorochrome, but will be of a higher energy (shorter
wavelength) than the emission wavelength it produces (e.g., green excitation wavelengths can produce
red emission wavelengths).
Fluorescent filters include three basic components: the excitation filter, the dichroic mirror, and
the emission filter (see Fig. 3). The excitation filter and dichroic mirror work together to produce
precise excitation wavelengths and to direct the light down into the objective lens and onto the specimen.
The resulting emission wavelengths then travel up through the objective and pass through the
mirror and emission filter so that precise wavelengths reach the eyes of the microscopist or camera.
Protocols for the various staining techniques will specify the fluorochromes and filters required
for their specific use.
Viewing Fluorochromes at the Microscope
The best fluorescent stain and filter combination for specimens stained with more than one fluorochrome
is one in which the color of each fluorochrome contrasts strongly against all other fluorochromes
present.
Single-emission fluorescence filters limit the wavelengths of light so that only one fluorochrome
is visible at a time. Dual- and triple-emission filters allow multiple fluorochromes to be seen at the
same time.