SUBMITTED BY: AKANKSHA

SUBMITTED TO : Dr. MANISHA

KUMARI
DEPARTMENT OF FORENSIC
SCIENCE
ROLL.NO:-3103
DATE OF SUBMISSION:-21/10/2023
M.Sc FORENSIC SCIENCE 1st Sem.(2023-
25)
 CONTENT
INTRODUCTION
PRINCIPLE OF FLUORESCENCE MICROSCOPE
COMPONENTS
WORKING
TYPES
APPLICATION
ADVANTAGES & DISADVANTAGES
CONCLUSION
REFERENCE
 INTRODUCTION
- Fluorescence Microscope: 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 FIGURE SHOWING
fluorescence microscope was devised in the early part of the FLUORSECENCE
twentieth century by August Köhler, Carl Reichert, and MICROSCOPE
Heinrich Lehmann, among others.
 Principle of Fluorescence
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 fluorophore.
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.
 COMPONENTS OF
FLUOROSCENT 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.
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
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.
 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 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.
 TYPES OF FLUORSCENT
MICROSCOPE
1.WiDe-field fluorescence microscopy
2.Confocal Fluorescence Microscopy
3.Total internal reflection fluorescence
microscopy (TIRFM).
 WIDE-FIELD FLUORSCENCE
MICROSCOPY
The basic FM is wide-field (WF) fluorescence microscopy,which is
excellent for 2D images of specimens, and the entire field can be
captured at once.For this microscope, a parallel beam of light
illuminates the whole specimen at once to excite the fluorophore. All
the resulting fluorescence of specimens can be viwed simultaneously,
allowing simple and fast imaging. For multiple-prob specimen, all the
Fluorescence can be viewed at once. Due to all parts of the specimen
can be viewed at once, it allows a quick selection of fluorescent cells to
image.
 LIMITATION AND RECENT
ADVANCEMENTS
The wide-field illumination and view not only focus information from the
corresponding section of the specimen but also allows out-of-focus light to arrive
camera, leading to low contrast and spatial resolution .

Recent improvement :- By using spatially structured illumination, the resolution of WF

microscopy can be increase. Gustafsson et al. [4] applied structured illumination
microscopy in 3D and doubled the 3D resolution in WF microscope. Xue et al. applied
Computational Miniature Mesoscope (CM2) to the wide-field microscope and
successfully presented 3D imaging
 Confocal Fluorescence Microscopy
Confocal microscopy is an advanced FM that can show
3D images with higher resolution than WF
fluorescence microscopy. Confocal microscopy can
eliminate out-of focus light from images and allow
thick specimens to be viewed with high resolution .
The best resolution that a confocal microscope can
attain is about 200nm . The major difference of
confocal microscopy in the region of illumination and
detection. A confocal microscope only illuminated a
diffraction-limited region of the specimen at one time
and only accepted a signal from that region .
TYPES:-1. Laser scanning confocal microscopy
2. Spinning disk confocal microscope
 LIMITATION AND ADVANCEMENT
Confocal microscope blocks out-of focus light by keep pinhole small, but much in-
focus light is also discarded.
2. The x-axis resolution in confocal microscopy is worse than the WF microscope.
3.Another consideration is the image speed,with confocal microscopy, an additional
factor can be the speed of the laser raster scan of the specimen. Due to inertia, the
speed of scan mirrors is limited.
4.The higher price is another shortage. Generally, confocal microscopy costs 2-7 times
more than WF microscopy.

ADVANCEMENT
To increase the resolution of confocal microscopy, Airyscan technology is applied.
Airyscan has a 32- channel detector array that can reassign pixels and summate images
from all detectors. This system provides 1.7 times higher resolution in the x-, y- and zaxis.
 Total Internal Refraction Microscopy
ATotal Internal Refraction Microscopy (TIRFM) is based on the excitation of
fluorophores by evanescent wave or field when the laser beam internally refracts.
Total internal refraction (TIRF) is an optical phenomenon that the light will reflect
instead of refracting when the light travels from a medium of high refractive index to
that of low refractive index, and the incident angle should be greater than the critical
angle.
In the case of TIRF, reflected light turns
into an electromagnetic field at the
interface, and an evanescent field forms
through the media of low refractive index,
which amplitude decays exponentially .
Therefore, the penetration depth of the
evanescent field can only be around 100
nm
 ADVANTAGES,LIMITATION & ADVANCEMENTS
Advantages:-Because of the low penetration depth of evanescent wave (usually less
than 100nm), there is almost no background fluorescence, causing super z-axial
resolution .
In addition, since only a small portion of the specimen is exposed to evanescent waves,
the effect of toxic gas and phototoxicity drastically weakened, extending experiment
time.
Limitation:-Because the energy of the evanescent wave decreases exponentially from
the interface, the fluorescence signal is not only strictly limited near the total reflection
interface (generally the interface between the glass slide and the sample, in the range
around 100 nm) but also too weak to detect.
Recent improvement:-TIRF is used in combination with many other microscopes to
compensate for its shortcomings and achieve better results.In recent research, fast two-
dimensional total internal reflection fluorescence, high-resolved structured illumination
microscopy (SIM), and traction force microscopy (TFM) are combined for higher spatial
and temporal imaging.Also, the combination of fast high-resolution spinning disk and
total internal reflection fluorescence microscopy .
 CONCLUSION
Although extensively adopted for a long time in cellular biology, FM
still has challenges. High cost, type of fluorochrome, and high noise
level, and other limitations are worth exploring. Because different
specimens need specific fluorochrome, we propose investigating how
to get or design proper fluorochrome to specimen efficiently.
Meanwhile, noise level always deteriorates results of algorithms,
which is shown in the combination of CMOS and SMLM. In past years,
the most adopted way to overcome limitations is to combine
different microscopes or cameras to achieve better results. In this
way, prospective research can try different combinations for greater
images. For example, researchers can combine low-cost with other
microscopes, producing low-cost and high-resolution images.
 REFERENCE
[1] Giepmans, B. N., Adams, S. R., Ellisman, M. H., &
Tsien, R. Y. (2006). The fluorescent toolbox for
assessing protein location and function. science, 312(5771), 217-224.
[2] Palmer, A. E., & Tsien, R. Y. (2006). Measuring
calcium signaling using genetically targetable
fluorescent indicators. Nature protocols, 1(3), 1057.
[3] Sanderson, M. J., Smith, I., Parker, I., & Bootman,
M. D. (2014). Fluorescence microscopy. Cold Spring Harbor Protocols, 2014(10), pdb-
top071795.
[4] Gustafsson, M. G., Shao, L., Carlton, P. M., Wang, . R., Golubovskaya, I. N., Cande,
W. Z., ... &
Sedat, J. W. (2008). Three-dimensional resolution doubling in wide-field
fluorescence microscopy by structured illumination. Biophysical journal, 94(12),
4957-4970.
[5] Hell, S. W. (2003). Toward fluorescence
nanoscopy. Nature biotechnology, 21(11), 1347-1355.
[6] Pawley, J. (Ed.). (2006). Handbook of biological
confocal microscopy (Vol. 236). Springer Science & Business Media.