SCHOOL OF BASIC AND APPLIED SCIENCES

Course Code :Q1PQ102T Course Name: ADVANCED PHYSICAL INSTRUMENTATION

FLUORESCENCE
MICROSCOPY
Name : Program Name:
SONAKSHI TYAGI (23SBAS2010046) M.Sc. FORENSIC SCIENCE
SHRUTI CHAUHAN(23SBAS2010048) Ist SEM
 BASIS OF FLUORESCENCE

 Fluorescence is the emission of photons by atoms or molecules whose electrons are stimulated to a higher excitation state
by radiant energy from an outside source.

 The principle of fluorescence microscopy also lies on the same.

The illumination light is
Becomes unstable, looses In the process , the
A fluorescent molecule separated from the weaker
It gets excited to a higher energy and almost molecule can release the
absorbs a photon of the emitted fluorescence
energy state immediately collapses back absorbed energy as a
appropriate wavelength through the use of spectral
to its initial ground state fluorescent photon.
emission filter.

 Molecules that are capable of fluorescing are called fluorescent molecules, fluorescent dyes, or fluorochromes.

 If a fluorochrome is conjugated to a large macromolecule (through a chemical reaction or by simple adsorption), the
tagged macromolecule is said to contain a fluorophore.
 FLUORESCENCE MICROSCOPE

 Fluorescence microscopy uses fluorescence and

phosphorescence to examine the structural organization,
spatial distribution of samples.

 Fluorescence microscopy images helps to study substances

present in low concentrations where high-sensitivity is
crucial to detect them.

 There are two main features that sets fluorescent microscope

apart from the traditional microscope - one is the type of
light source and the other is the use of specialized filter
elements.
 PARTS OF FLUORESCENCE MICROSCOPE

 A powerful light source (xenon or mercury arch lamp):

o The light emitted from the mercury arc lamp is 10-100 times brighter
than most incandescent lamps and provides light in a wide range of
wavelengths, from ultra-violet to the infrared.

 Excitation filter:
o The purpose of the excitation filter is to filter out all wavelengths of the light
source, except for the excitation range of the fluorophore under inspection.

 The Dichroic mirror (beam splitter):

o The Dichroic mirror or beam splitter is placed at an angle of 45° between
the excitation filter and emission filter.
o The function of a dichroic filter is to reflect the excitation signal towards
the fluorophore and to transmit the emission signal towards the detector.
 PARTS OF FLUORESCENCE MICROSCOPE

 Emission filter:
o The emission filter is located within the imaging path of a
fluorescence microscope.
o Its job is to filter out the entire excitation range and to transmit the
emission range of the fluorophore under inspection.

 Objective lens:
o The purpose of the objective lens is to transmit light to the
sample to form the image.
o The light passes down through the dichroic mirror before
 Camera system: reaching the objective lens.
o A camera system helps to record the images of the specimen
with high-resolution.
 HOW FLUORESCENCE MICROSCOPY WORKS

When the atom absorb photon from the outer source, it gets excited and jumps to a higher energy state

Instantly the unstable atom losses energy and return to the lower energy state, releasing the extra energy in the
form of fluorescence..

But this absorption and emission of energy does not stay continuous. It changes with the time creating a shift
in the absorption peak and emission peak

This difference in peak is known as stroke shift

Absorption and excitation spectra are distinct but usually overlap, sometimes to the extent that they are nearly
indistinguishable.

The widths and locations of the spectral curves are important, particularly when selecting two or more
fluorochromes for labeling different molecules within the same specimen.
FLUORESCENCE MICROSCOPY

Normalized absorption and fluorescence emission spectra of fluorescein-

conjugated IgG. The difference in nanometers between the excitation and
emission maxima is called the Stokes shift.
FLUORESCENCE MICROSCOPY

Phalloidin dye is
used to stain actin
fibers in
mammalian cells
DAPI is a
fluorescent stain
that binds strongly
to A-T rich regions
in DNA
Hoechst stain are
part of a family of
blue fluorescent
dyes used to stain
DNA
 LIMITATIONS

Interference
Low light
Not suitable with
levels
for certain membrane
Photo Low light
Phototoxicity Limited samples systems
bleaching levels and the
Cells can be spatial Fluorescence The addition of
Fluorophores resolution fading of
susceptible to microscopy dyes to a
can lose their fluorescent
phototoxicity, The resolution may not be membrane
ability to specimens are
especially of fluorescence suitable for system can
fluoresce when two of the
when using microscopy is imaging certain potentially
illuminated, a primary
short- limited by the types of interfere with
process called difficulties to
wavelength wavelength of samples, such the properties
photobleaching overcome in
light. light used. as highly of the
. fluorescence
autofluorescent liposomal
photomicrogra
tissues. delivery
phy.
system.
SIGNIFICANCE IN FORENSIC SCIENCE

• Fiber testing • Detection of

teeth and
bones

Examining Examination
latent of soil
fingerprints samples

Examination
Examination
of
of gunshot
questioned
residue
document
• Examination • Examination
of of semen and
archeological seminal
objects stains
 SOURCES & REFERENCES

Fundamentals of light microscopy and electronic imaging by Douglas B. Murphy

https://microscopeinternational.com/fluorescence-microscopy/#

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711767/

https://www.google.com/

https://link.springer.com/chapter/10.1007/0-306-47057-8_7

https://www.sciencedirect.com/science/article/pii/007961079190013I