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1. Fluorescence microscopy
Mandira S. Bhosale
MSc part 1 sem 2
FMBCH2122005

2. History:
• Fluorescence was first discovered in 1845 by Fredrick W. Herschel.

3. History:
• British scientist Sir George G. Stokes further studied and discovered,
that fluorescence emission from an object represents a longer
wavelength than the UV light that originally excited the object.
• He coined the term Fluorescence.
• Hence , the shift from shorter
wavelength to the higher
wavelength known as Stokes shift.

4. History :
• In the early 1900s, the first uses of fluorophores in biological
investigations were performed to stain tissues, bacteria, and other
pathogens.
• This was later developed into fluorescence microscopy by Carl Zeisi
and Carl Reichert.
• Fluorescence labeling was achieved by Ellinger and Hirt in the early
1940s.
• The cloning of green fluorescent protein (GFP) was achieved in the
early 1990s first and was easily applied to fluorescence microscopy.

5. Fluorescence microscopy:
• Fluorescence :
Fluorescence is the emission of light by a substance
that has absorbed light or other
electromagnetic radiation.
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• Microscopy :
Microscopy is the technical field of using microscopes
to view objects and areas of objects that cannot
be seen with the naked eye.

6. • Fluorescence microscopy is study in which microbes or molecules
observed under microscope with the help of fluorescence.
• A fluorescence microscope is an optical microscope that
uses fluorescence instead of, or in addition to, scattering, reflection,
and attenuation or absorption, to study the properties of organic
or inorganic substances.
• Fluorescence microscopy uses fluorescence to examine the structural
organization, spatial distribution of samples.
• It is particularly used to study samples that are complex and cannot be
examined under conventional transmitted-light microscope.

8. Fluorescence microscope Vs light microscope:
• Light microscopes use light in the 400-700nm range, the range through which
light is visible to the human eye ,but fluorescence microscopy uses much higher
intensity light.
• light microscopy uses visible light, the resolution is more limited. Fluorescence
microscopy, uses light produced by the fluorophores in the sample.
• light microscopes do still have a prominent place in microscopy however, both in
combination with dyes and sometimes without.
• Fluorescence microscopy is used in conjunction with other kinds of light
microscopy because it creates images from reflected light, rather than direct light.

9. Principle:
• Most cellular components are colorless and hence cannot be visualize under a
normal light microscope.
• Hence they are stained with fluorescent dye , also known as fluorophores or
fluorochromes.
• These molecules stains that components and absorbs the excited light of
shorter wavelength and emits the light of longer wavelength.
• This emitted light is called as fluorescent light.
• Emitted fluorescent light produces an image of desired part of molecule and
which then projected on computer screen.
• Or can be observed on microscope with naked eyes.

10. Components:

11. • Light source :
i. 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.
ii. Lasers are mostly used for complex
fluorescence microscopy techniques, while
xenon lamps, and mercury lamps, and LEDs
are commonly used for wide-field &
epifluorescence microscopes.

12. • 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.
• Excitation filter and emission filter:
i. The exciter is 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.
ii. 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.

13. • Detector :
Detector analyze the emitted light it has
electron imaging sensor and projects the final
image of specimen on computer screen.
• Darkfield condenser:
It provides a black background against which
the fluorescent objects glow.

14. • Preparation of
specimen:
1. Fluorescent dyes
2. Tagging of proteins
3. Immunofluorescence

15. • Light shorter wavelength of lights (UV rays or blue light) generated
from mercury vapor arc lamp passes through the excitation filter
• Which allows only the short wavelength of light to pass through and
removes all other non-specific wavelengths of light.
• The filtered light is reflected by the dichroic filter and falls on the
sample (Fluorophore-labeled).
• The fluorochrome absorbs shorter wavelength rays and emits rays of
longer wavelength (lower energy) that passes through the emission
filter.
• The emission filter blocks (suppresses) any residual excitation light
and passes the desired longer emission wavelengths to the detector.
• Thus the microscope forms glowing images of the fluorochrome-
labeled microorganisms against a dark background.

16. Examples:
• Fluorescent image of human stem cells stained
with monoclonal antibodies markers under the
microscopy nuclei in blue and actin
microfilaments in green
• English daisy flower ,cross section through a
flower head, fluorescence microscope image
• Blue stained DNA.

17. Types:
• Epifluorescence microscopes:
The most common type of fluorescence microscope in
which, 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.

18. • Multiphoton microscope:
In this type of microscope, multiphoton fluorescence
excitation results in the capture of high-resolution three-
dimensional images of specimen 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.

19. Advances in
microscopy

20. • Widefield microscopy • Lightsheet microscopy • Stochastic optical
reconstruction
microscopy
• Electron cryo
microscopy

21. • Fluorescence recovery after photobleaching (FRAP)
• Fluorescence correlation spectroscopy (FCS)
• Super resolution stimulated emission depletion (STED)
• Photoactivated localization microscopy (PALM)
• Structured illumination microscopy (SIM) combined with
TIRF
• Deep imaging via emission recovery (DIVER)
• Nanoscale precise imaging by rapid beam oscillation
(nSPIRO)

22. • On 8 October 2014 , the Nobel
prize in chemistry was awarded to
Eric Betzig, William Moerner and
Stefan Hell for the development of
super resolved fluorescence
microscopy

23. Advantages:
• Fluorescence microscopy is the most popular method for studying the
dynamic behavior exhibited in live-cell imaging.
• Isolation of individual proteins with a high degree of specificity can be
visualized.
• Different molecules can be stained with different colors, allowing
multiple types of the molecule to be tracked simultaneously.
• These factors combine to give fluorescence microscopy a clear
advantage over other optical imaging techniques, for both in vitro and
in vivo imaging.

24. Limitations:
• Fluorophores lose their ability to fluoresce as they are illuminated in a
process called photobleaching..
• Cells are susceptible to phototoxicity, particularly with short-
wavelength light.
• Unlike transmitted and reflected light microscopy techniques
fluorescence microscopy only allows observation of the specific
structures which have been labeled for fluorescence.

25. Applications:
• Fluorescence Microscopy used for Cell Labeling
• Protein Characterization achieved by Fluorescence Microscopy.
• Fluorescence Microscopy in forensic field for latent fingerprints, GSR.
• Detection of molecular interaction e.g . Proteins, DNA
• Conformational changes , structural studies of molecules.
• Tissue characterization by auto fluorescence.
• Visualization of molecules with immunofluorescence.
• Characterization of new materials.

26. References:
• https://microscopeinternational.com/fluorescence-microscopy/
• https://microbeonline.com/fluorescence-microscope-principle-types-applications/
• https://pubmed.ncbi.nlm.nih.gov/23585290/
• https://allmymedicine.com/health-news/the-history-of-fluorescence-microscopy/
• http://www.scholarpedia.org/article/Fluorescent_proteins
• https://microbenotes.com/fluorescence-microscope-principle-instrumentation-applications-
advantages-limitations/
• https://microscopeinternational.com/widefield-fluorescence-microscopy/
• https://microscopeinternational.com/total-internal-reflection-fluorescence-microscopy/
• https://www.nobelprize.org/educational_games/physics/microscopes/fluorescence/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835776/
• https://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22029
• https://di.uq.edu.au/community-and-alumni/sparq-ed/cell-and-molecular-biology-
experiences/immunofluorescence/background-immunofluorescence

27. Thank you!!