What is Fluorescence Microscopy?

A fluorescence microscope is an optical microscope that employs fluorescence for examining specific
properties of select organic and inorganic substances. A fluorescence microscope functions by
observing labeled samples; the labels here are the specific fluorescent molecules (or clusters of
molecules)—termed fluorophores—that emit light when stimulated at specific frequencies.

Components of a fluorescence microscope include a light source (typically high-intensity lamps, lasers,
or LEDs), excitation filters, a dichroic mirror, and emission filters. The light source provides the
stimulation light needed to view a specimen. The excitation and emission filters allow for wavelength
specificity. The dichroic mirror directs the stimulation light onto the specimen (it reflects it onto the
specimen) but allows the emitted light to filter through to the detector.

Why is fluorescence microscopy such a universally important technique in so many scientific

fields?
Because it enables selective tagging and the ability to visualize exactly what’s going on in the chaotic
assembly that is a biological entity with so many moving features. For instance, if the molecule of
interest can be conjugated to a fluorophore, one can determine where it exists in context to the cell, how
it moves with respect to other entities, how it changes shape over time and space due to specific
stimulation—none of which would be able to be assessed without conventional microscopy.
Fluorescence microscopy has applications across the board. For instance, fluorescence microscopy is
used in the analysis of diagnostic antibodies and antigen expression in tissue sections in biomedical
research, as well as localization studies for proteins and nucleic acids in certain parts of the cell. Less
extensively, microbiology, neuroscience, and developmental biology rely on fluorescence microscopy
to better understand localization and organizational phenomena.
The history of fluorescence microscopy spans more than a century. For example, in 1852, British
scientist Sir George G. Stokes officially identified fluorescence and observed the Stokes shift (the
change in emission spectrum to longer wavelengths). In the twentieth century, August Köhler and Carl
Reichert observed fluorescence while using optical microscopes and, a bit later, ultraviolet microscopes
(albeit initially as an undesired feature). Yet throughout the following decades, subsequent
developments in fluorescent dyes and alternative light sources validated fluorescence microscopy as a
lasting path for scientific exploration.

Types of Fluorescence Microscopes

There are many forms of fluorescence microscopy as some transpire in different years and some have
different advantages. These include:
1. Wide-Field Epifluorescence Microscopy- the most standard form of fluorescence
microscopy. This technique uses a uniform approach to excite the entire specimen at the
excitation wavelength, and those regions of the specimen that fluoresce accept the initial
wavelength and re-emit at the emission wavelength.
 2. Confocal Fluorescence Microscopy—Uses point illumination and spatial pinholes to
eliminate out of focus light during imaging. Produces optical sectioning as the image is
reconstructed by scanning the sample in a step-wise fashion. Increased resolution and
contrast by using for more substantial samples.
3. Total Internal Reflection Fluorescence (TIRF) Microscopy- TIRF excites
fluorophores in a very specific plane relative to the glass-water interface, on the order of
~100 nm from the cell membrane. Therefore, this technique is good for studying
anything at the cell surface and effective for membrane dynamics and cell-substrate
interactions.
4. Two Photon (Multiphoton) Microscopy- Two photon microscopy utilizes near-
infrared light which only excites fluorophores at the focal point; therefore, antagonistic
photodamage is minimized and one can see deeper into live tissue. This technique is
applicable for intravital imaging of dynamic processes.
5. Fluorescence-Lifetime Imaging Microscopy (FLIM) – FLIM measures the decay time
of emitted fluorescence of fluorophores, rendering information about the localization of
the fluorophores’ environment (pH, ion concentrations). Therefore, it helps determine
compound interactions and the overall biochemical state of the cells.

Principle of Fluorescence Microscope – How does fluorescence microscopy

work?
The fluorescence microscope is based on fluorescence. The sample’s molecules of interest are stained
with fluorescent dyes (or fluorophores), and upon exposure to light of one wavelength, the
fluorophore absorbs energy and emits it at a longer wavelength. The emitted light that passes through
is combined to create the image of the sample. The first step in this entire procedure is specimen
preparation with appropriate fluorescent stains. A light source (mercury vapor lamp or laser) generates
white light which passes through an excitation filter.
The excitation filter creates the specific wavelength needed for exciting certain fluorophores present in
the specimen. Next, the light travels through a dichroic mirror, which upon receiving the wavelength
from the excitation filter, reflects it downwards toward the specimen through the objective lens. The
excitation light strikes the specimen, exciting the fluorophores which then emit wavelengths of light at
longer colors. This occurs in reverse fashion as the light travels back up through the objective lens to
the dichroic mirror.
The dichroic mirror allows the longer wavelength emitted light to travel up and continue while reflecting
excess excitation wavelength light back downward. After the dichroic mirror, however, the emitted light
strikes an emission filter which serves to further discriminate the longer excitation wavelengths so that
only emitted wavelengths driven by fluorophores can be seen in the eyepiece or detector. Thus, the
result is a brightly colored fluorescent specimen against a dark background.
The fact that researchers can obtain such resolution and differentiation means that they are essentially
creating their own version of reality from what is essentially biological white noise. By marking
particular molecules with the studied fluorophores, they discover where those molecules reside, how
they interact with others and themselves over time—and in real time—acquiring information that would
otherwise go unnoticed by the human eye and standard microscopy.
Parts of Fluorescence Microscope
A fluorescence microscope is comprised of:
1. Light Source- the element that generates the excitation light which stimulates the
fluorescent particles within the specimen. The more typical illumination sources found in
fluorescence microscopes are high-intensity lamps (mercury-vapor lamp, xenon arc
lamp), lasers, and high-intensity LEDs.
2. Excitation Filter- This filtering element selects the precise wavelength of light required
to produce fluorescence to stimulate the fluorescent tags.
3. Dichroic Mirror (Beamsplitter)- A specialized mirror reflecting excitation light
directed back to the specimen yet allows emission fluoresced light access to the imaging
device.
4. Objective Lens – Captures both excitation light received by the specimen and fluoresced
emitted light given off by the specimen, thus factoring into the composite image.
5. Emission Filter– Located between the dichroic mirror and the emission viewer
(observer), it allows fluoresced emitted light of certain wavelengths only to pass through
while filtering out any superfluous excitation light.
6. Detector- Accepts the given off fluorescent light for image formation. Detectors are
typical eyepieces for viewing and digital cameras for imaging.
Sample Preparation for Fluorescence Microscope
There is a general sample preparation procedure for fluorescence microscopy, which can be modified
according to the type of sample and what one intends to view. The general sample preparation
involves:
1. Fixation. This is when morphology is preserved and localization of structures is
preserved. Generally, chemical fixatives are applied—paraformaldehyde or
glutaraldehyde—which cross-link proteins to preserve the spatially arranged features of
that cell type. Fixation is necessary to preserve the sample for proper treatment and
subsequent imaging.
2. Permeabilization—Another aspect after fixation is permeabilization of the cells so that
the now previously inaccessible intracellular antigens can be rendered available to
fluorescent probes or antibodies. Detergents like Triton X-100 or saponin permeabilize
the cells by effectively punching holes in the membrane of the cell while still maintaining
its overall structure.
3. Blocking—Another necessary component to successful immunohistochemistry is the
necessity of a blocking step to reduce non-specific binding of fluorescent probes or
antibodies. A blocking solution of a cocktail of proteins is generated and applied to the
samples. Nonspecific fluorescence is reduced with many proteins such as bovine serum
albumin (BSA) while specific binding of the staining is increased.
4. Staining/Labeling- The specimen is stained/labelled with fluorescent dyes or antibodies
that stick to the molecules or structures of interest. This can be a direct label (primary
antibodies conjugated to fluorophores) or indirect (primary antibodies for the antigen of
interest and secondary labelled antibodies) based on what’s needed for the experiment.
5. Mounting- The mounting happens at the very end when the specimen is placed on a
slide with a mounting medium that usually contains antifade agents to preserve the
fluorescence. A coverslip is added for protection and to facilitate the correct viewing
optics.

Light Path In Fluorescence Microscopy

1. First, the light source for the microscope is a high-intensity mercury lamp. The lamp generates
white light in the visible spectrum.
2. Second, an exciter filter is used to let blue light pass through (filtering out all other wavelengths).
Therefore, it creates the excitation light required by the specimen.
3. Third, there is a dichroic mirror in the pathway of the light. It reflects blue light downward to
the specimen but lets other wavelengths—like that which is emitted from the fluorescent stain—
continue on their path.
4. Next, the blue light hits the sample, which has been stained with a fluorescent dye. The dye
takes in the blue light and fluoresces a different color, often green. The resultant green light
travels back up and hits the dichroic mirror, which only accepts that wavelength for passage.
5. Next, the emitted light hits the barrier filter. This filter blocks any remaining blue light from
escaping and allows the green light to pass through.
6. Ultimately, the observer sees the stained areas of the sample glowing green on a black
background. The unstained areas are clear since they do not fluoresce.
Types of Fluorescence microscope light source
The light source of a fluorescence microscope is an important component that is responsible for
providing the excitation light that is used to excite the fluorophores in the sample.
There are several types of light sources that are commonly used in fluorescence microscopy,
including:
1. Mercury arc lamps- Mercury arc lamps are one of the most commonly used light
sources in fluorescence microscopy. They produce a broad spectrum of light that can be
used to excite a wide range of fluorophores. However, they may produce unwanted
emissions, which can interfere with fluorescence imaging.
2. Metal halide lamps – Metal halide lamps are a newer type of light source that produce a
more focused spectrum of light than mercury arc lamps. They are often used to excite
specific fluorophores and can provide higher intensity and more stable light output than
mercury arc lamps.
3. LED light sources– LED (light-emitting diode) light sources are becoming increasingly
popular in fluorescence microscopy due to their long lifetimes and low power
consumption. They produce a narrow spectrum of light that can be used to excite specific
fluorophores and can be easily controlled and modulated for advanced imaging
applications.
4. Laser light sources- Laser light sources produce a highly focused and coherent beam of
light that can be used to excite specific fluorophores. They are often used in advanced
imaging techniques, such as superresolution microscopy, that require high-intensity,
highly focused light.

Analyzing a Specimen With a Fluorescence Microscope

• First, fluorescence microscopy needs a lot of preparation for the sample. The hardest
part is that the structures of interest need to be fluorescent. If they are not, they need
to be stained through some specific labeling process, some of which are fluorescent
dyes or the incorporation of fluorescent proteins.
• Second, the fluorescent labeling occurs. This can happen in a few ways: Fluorescent
stains of biological origin can bind to specific biological molecules. For instance,
there are nucleic acid stains like DAPI and Hoechst, and phalloidin binds to actin
filaments. In immunofluorescence, antigens are identified by antibodies.
The antibody that recognizes the targeted antigen is the primary antibody; the
antibody that attaches to the primary is the secondary antibody, which is conjugated
to a fluorophore. In live/dead fluorescent protein experiments, fluorescent proteins
are utilized through genetic manipulation and subsequently, fluorescence dyeing and
coloring is observed. Ultimately, however, a microscope is set up.
• The optics of the microscope need to correspond with the chosen fluorophore.
Generally, the dye’s manufacturer supplies details to determine the appropriate filter
compatibility and correspondence.
 • Next, slide making. Decontaminate the slide and the coverslip to prevent any
autofluorescence. Place a drop of the sample on the slide and cover with the
coverslip; you’re ready to view the sample.
Start with low power and focus, adjusting light and position for the best view. Increase
magnification as desired, continuing to focus and adjust position for the best view.
• Last, saving images of anything worthwhile. Make sure to title images or at least
remember what you have so you can find it later.
• Then there’s photobleaching. Many fluorophores become photobleached from too much
excitation light exposure. Some versions are worse than others. The fixes are shorter,
more gentle exposures or finding a different dye with better stability. But ultimately,
where this happens is a good thing. Some forms of analysis—FRAP (fluorescence
recovery after photobleaching), for instance—need a region of interest to be
photobleached so the fluorescence recovery can assess diffusion rates of fluorophores in
that region.

Care of a Fluorescence Microscope

• Always turn off the microscope after use.
• Clean lenses with a soft, lint-free cloth.
• Use a blower brush to remove dust.
• Apply immersion oil only to the 100x objective.
• Remove oil promptly to prevent residue.
• Cover the microscope when not in use.
• Store in a dry, dust-free environment.
• Regularly check and replace light bulbs.
• Lubricate moving parts as recommended.
• Keep the stage clean to avoid interference.

• Inspect the condenser for cleanliness.

• Use lens cleaning solution sparingly.
• Avoid using abrasive materials on lenses.

• Handle the microscope with care to prevent misalignment.

• Refer to the user manual for specific maintenance guidelines.

Application of Fluorescence Microscope

• Used to observe fluorescently labeled samples.
• Helps study cell structures and proteins.
• Enables detection of specific molecules.
• Allows live-cell imaging in real time.