Fluorescence microscopy uses fluorescence and phosphorescence instead of reflection and absorption to study organic or inorganic substances. It works by illuminating a specimen with light that excites fluorescent molecules called fluorophores within the specimen. The fluorophores then emit light of a longer wavelength, which can be filtered and observed. Key components include a light source, excitation and emission filters, and fluorescent dyes or stains used to label structures of interest within specimens. It has many applications in fields like immunology, cell and molecular biology, and live/dead discrimination of microbes.

1. Fluorescence Microscopy

2. INTRODUCTION
 British scientist Sir George G. Stokes first described fluorescence in 1852.
 The fluorescence microscope was devised in the early part of the twentieth
century by August Kohler, Carl Reichert, and Heinrich Lehmann, among
others.
: A fluorescence microscope is an optical
microscope that uses fluorescence and phosphorescence instead of, or in
addition to, reflection and absorption to study properties of organic or
inorganic substances.
: A process by which a photon is absorbed at one
wavelength and released at a different wavelength or energy.
: Phosphorescence is a specific type of
photoluminescence related to fluorescence. Unlike fluorescence, a
phosphorescent material does not immediately re-emit the radiation it
absorbs.

3. PRINCIPLE
 The specimen is illuminated with light of a specific wavelength.
 Which is absorbed by the fluorophores, causing them to emit light of
longer wavelengths (i.e., of a Principle different color than the absorbed
light).
 The illumination light is separated from the much weaker emitted
fluorescence through the use of a spectral emission filter.
 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.

4. Partsof Fluorescence Microscope
1. 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.
2. 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.

5. Partsof Fluorescence Microscope
3. 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.
4. 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.
5. 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.

6. Specimenpreparation
1.Wash the cells twice and use tweezers to carefully place the coverslip with upturned
cells into the humidified chamber.
2.Fix with 4 % formaldehyde for 10 minutes and wash 3 ×.
3.Permeabilize with 0.1 % TX-100/PBS for 15–20 minutes and wash 3 ×.
4.Block with 5 % normal goat serum/PBS or 1 % BSA/PBS for 45 minutes (no washing
required).
5.Dilute the primary antibody in blocking solution and apply it for 2 h (or overnight at
4 °C). Wash 4 × thoroughly to remove unbound primary antibody.
6.Incubate with the secondary antibody for 1 h, diluted in blocking solution or wash buffer.
7.Aspirate the secondary antibody and, if desired, incubate with Hoechst or DAPI
[1 µg/mL] in PBS for 10 minutes. Wash 4 × thoroughly, even without nuclear staining.
8.Take the coverslip gently with tweezers and dip it into dH2O to remove residual salts of
the wash buffer.
9.Provide a drop of mounting medium on a microscope slide and lay the coverslip with the
cells upside down on this drop. Press the specimen with the tweezers slightly so that the
mounting medium is well distributed, without squeezing the sample.
10.The preparation is ready for microscopy after curing.

7. Howto Use a Fluorescence Microscope
Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Step 7
Locate the light
switch on the side of
the microscope that
turns on the light.
Turn the microscope
on. Write down the
exact time you turn
on the light. The
florescent light is
mercury-based, and
a time log must be
kept for exposure
and use of the light.
Locate the toggle
switch on the right
side of the
microscope between
the oculars and
objectives. This
switch controls the
shutter for the
mercury light to the
objective lens.
Select the
appropriate dye for
your object (this will
depend entirely on
what you are going
to be studying). The
most common dyes
include I3 (for use
with CTC, DTAF and
fluorescein), A (for
use with DAPI and
f420), N21 (for use
with Rhoda mine)
and L3
Put the filter (dye)
into the tray
operated by the
silver sliding knob.
To remove the tray,
simply pull the silver
knob out.
Select the lens you
would like to use.
The 63x objective
lens will have the
highest numerical
aperture. The 100x
objective lens will
have the highest
magnitude that can
be used with the
mercury-based
florescent light
source.
Turn the light off
when finished, and
mark the time. Wait
30 minutes before
turning the light back
on, or the lamp could
explode. It is a good
idea to keep track of
how many hours the
lamp is in use and
replace it according
to the manufacture's
guidelines.
Clean off the
microscope lens with
lens paper, or if
really dirty, use a
cotton swap and
glass cleaner.

8. applicationsof Fluorescence Microscopy
1. Immunology: An antibody is first prepared by having a fluorescent chemical
group attached, and the sites (e.g., on a microscopic specimen) where the antibody
has bound can be seen, and even quantified, by the fluorescence.
2. Cell and molecular biology:
 detection of colocalization using fluorescence-labelled.
 Imaging structural components of small specimens, such as cells.
 Viewing specific cells within a larger population with techniques such as FISH.
Detection and determination of the proteins localization in cell and tissue
3.Membrane potential : Bacterial membrane potential may be analyzed using DiOC2, which exhibits green
fluorescence in all bacterial cells, but shifts to red fluorescence as the dye becomes more concentrated in cells with
larger membrane potentials. Mitochondrial membrane potential may be analyzed in the same manner with JC-1.
4. Live/dead bacteria discrimination : You can test how fast an antibiotic is killing microbes: live cells have intact
membranes and are impermeable to dyes such as propidium iodide, which only leaks into cells with compromised
membranes. Thiazole orange enters all cells, live and dead, to varying degrees. Thus a combination of these two dyes
provides a rapid and reliable method for discriminating live and dead bacteria.

9. advantagesof Fluorescence Microscopy
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.
limitationsof Fluorescence Microscopy
1. 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.
2. 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.
3. Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of
the specific structures which have been labeled for fluorescence.