Fluorescence microscope

Introduction
Fluorescence is a property of some substances to excite emission of visible light by the
absorption of invisible ultraviolet radiations. In general the wavelength of the emitted light is
longer than the wavelength of the absorbed light. Fluorescence microscopy uses this property to
specifically image materials of interest.

Some materials are auto fluorescent(e.g. : the amino acid tryptophan fluoresces in the 250 to
400 nm wavelength region), whereas others can be made to fluoresce by treatment with
fluorescent chemicals called fluorochromes .(e.g. : Fluorescein isothiocyanate [FITC] absorbs blue
light and emit green light, Rhodamine absorb green light and emit red light. These compounds are
used in immuno-fluorescence microscopy.)

Principle
The optical principle of fluorescent microscope is similar to that of a compound microscope
except for the incorporation of two filters to the optic system (source filter and complementary
filter).
Compound microscope has two sets of lenses, the objective and the eyepiece.
When the beam of light passes through the object and then convex lenses of objective it forms a
real inverted and enlarged image of the object in the focal plane of eyepiece(by adjustment) This
image now act as object for the eyepiece.

Working
Equipment includes:
1) A source of ultraviolet rays
2) Suitable filters for isolating the radiations (source filter and complementary filter.)
3) A standard microscope.
On illumination by ultraviolet radiation, many substances are capable of absorbing light of
different wavelengths. This property is called fluorescence and the substances are called
fluorescent substances.
Filters are used for both the incident light and the scattered light so that only a narrow band
of wavelength is passed in each case.
Among the two filters, the source filter is placed between the source of light and mirror and
the complementary filter in front of the ocular lens in the body tube. The source filter transmits the
ultraviolet rays towards the object i.e.; allows only those wavelengths that will be absorbed to
impinge on the specimen, and the complementary filter absorbs the rays, which are not absorbed
by the object.ie; allows only the emitted wavelengths to pass through. Since the emitted
wavelengths are different from the absorbed wavelength, all background is filtered out and only
the image of the sample is allowed through.

Application
1) Examination of acid-fast bacteria (M. tuberculosis).
2) Differentiation of living and dead micro-organisms.
3) Examination of hair for mold spores.
4) Location of chemotherapeutic agents in tissues.
5) To localize the antigens for a number of viruses.