Fluorescence Microscopy- Definition, Principle, Parts, Uses

A fluorescence microscope is an optical microscope that uses

fluorescence and phosphorescence instead of, or in addition to,
reflection and absorption to study the properties of organic or
inorganic substances. Fluorescence is the emission of light by a
substance that has absorbed light or other electromagnetic
radiation while phosphorescence is a specific type of
photoluminescence related to fluorescence. Unlike fluorescence, a
phosphorescent material does not immediately re-emit the
radiation it absorbs. The fluorescence microscope was devised in
the early part of the twentieth century by August Köhler, Carl
Reichert, and Heinrich Lehmann, among others.

Principle of Fluorescence Microscope

Most cellular components are colorless and cannot be clearly
distinguished under a microscope. The basic premise of
fluorescence microscopy is to stain the components with dyes.
Fluorescent dyes, also known as fluorophores or fluorochromes,
are molecules that absorb excitation light at a given wavelength
(generally UV), and after a short delay emit light at a longer
wavelength. The delay between absorption and emission is
negligible, generally on the order of nanoseconds.
The emission light can then be filtered from the excitation light to
reveal the location of the fluorophores.
 Fluorescence microscopy uses a much higher intensity light to
illuminate the sample. This light excites fluorescence species in
the sample, which then emits light of a longer wavelength.
 The image produced is based on the second light source or the
emission wavelength of the fluorescent species rather than from
the light originally used to illuminate, and excite, the sample.
Working
Light of the excitation wavelength is focused on the specimen
through the objective lens. The fluorescence emitted by the
specimen is focused on the detector by the objective. Since most
of the excitation light is transmitted through the specimen, only
reflected excitatory light reaches the objective together with the
emitted light.
Forms
The “fluorescence microscope” refers to any microscope that
uses fluorescence to generate an image, whether it is a more
simple set up like an epifluorescence microscope, or a more
complicated design such as a confocal microscope, which uses
optical sectioning to get better resolution of the fluorescent
image.
 Most fluorescence microscopes in use are epifluorescence
microscopes, where excitation of the fluorophore and detection of
the fluorescence are done through the same light path (i.e.
through the objective).

Typical components of a fluorescence microscope are:

 Fluorescent dyes (Fluorophore)
 A fluorophore is a fluorescent chemical compound that can re-
emit light upon light excitation.
 Fluorophores typically contain several combined aromatic groups,
or plane or cyclic molecules with several π bonds.
 Many fluorescent stains have been designed for a range of
biological molecules.
 Some of these are small molecules that are intrinsically
fluorescent and bind a biological molecule of interest. Major
examples of these are nucleic acid stains like DAPI and Hoechst,
phalloidin which is used to stain actin fibers in mammalian cells.
 A light source
 Four main types of light sources are used, including xenon arc
lamps or mercury-vapor lamps with an excitation filter, lasers,
and high- power LEDs.
 Lasers are mostly used for complex fluorescence microscopy
techniques, while xenon lamps, and mercury lamps, and LEDs
with a dichroic excitation filter are commonly used for wide-field
epifluorescence microscopes.
 The excitation filter
 The exciter is typically a bandpass filter that passes only the
wavelengths absorbed by the fluorophore, thus minimizing the
excitation of other sources of fluorescence and blocking
excitation light in the fluorescence emission band.
 The dichroic mirror
 A dichroic filter or thin-film filter is a very accurate color filter
used to selectively pass light of a small range of colors while
reflecting other colors.
 The emission filter.
 The emitter is typically a bandpass filter that passes only the
wavelengths emitted by the fluorophore and blocks all undesired
light outside this band – especially the excitation light.
 By blocking unwanted excitation energy (including UV and IR) or
sample and system autofluorescence, optical filters ensure the
darkest background.

Types of Fluorescence Microscopes

There are various types of fluorescence microscopes. Some of the
common types are:

Epifluorescence microscopes
It is the most common type of fluorescence microscope. In this microscope,
excitation of the fluorophore and detection of the fluorescence are done
through the same light path (i.e., through the objective).

Confocal microscope
In this type of fluorescence microscope, high‐resolution imaging of thick
specimens (without physical sectioning) can be analyzed using fluorescent-
labeled dye.

Multiphoton microscope
In this type of microscope, multiphoton fluorescence excitation captures
high-resolution three-dimensional images of specimens tagged with highly
specific fluorophores.

Total internal reflection fluorescence (TIRF) microscope

Total internal reflection fluorescence microscopy (TIRFM) exploits the
unique properties of an induced evanescent wave or field in a limited
specimen region immediately adjacent to the interface between two media
having different refractive indices.

Applications of Fluorescence Microscope

 To identify structures in fixed and live biological samples.
 Fluorescence microscopy is a common tool for today’s life
science research because it allows the use of multicolor staining,
labeling of structures within cells, and the measurement of the
physiological state of a cell.
 Advantages of Fluorescence Microscope
1. Fluorescence microscopy is the most popular method for
studying the dynamic behavior exhibited in live-cell imaging.
2. This stems from its ability to isolate individual proteins with a
high degree of specificity amidst non-fluorescing material.
3. The sensitivity is high enough to detect as few as 50 molecules
per cubic micrometer.
4. Different molecules can now be stained with different colors,
allowing multiple types of the molecule to be tracked
simultaneously.
5. These factors combine to give fluorescence microscopy a clear
advantage over other optical imaging techniques, for both in
vitro and in vivo imaging.

Limitations of Fluorescence Microscope

 Fluorophores lose their ability to fluoresce as they are
illuminated in a process called photobleaching. Photobleaching
occurs as the fluorescent molecules accumulate chemical
damage from the electrons excited during fluorescence.
 Cells are susceptible to phototoxicity, particularly with short-
wavelength light. Furthermore, fluorescent molecules have a
tendency to generate reactive chemical species when under
illumination which enhances the phototoxic effect.
 Unlike transmitted and reflected light microscopy techniques
fluorescence microscopy only allows observation of the specific
structures which have been labeled for fluorescence.