Fluorescence Microscopy and Epifluorescence and application based research paper.

1. Fluorescence Microscopy
Saransh Khandelwal
Technical Superintendent,
IIT Hyderabad

2. Content
 Introduction
 Basics of Fluorescence
 Epi-Fluorescent Microscope
 Working
 Applications in Biology
 Advantages and Limitations
 References
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3. Introduction
Generation of luminescence through excitation of
a molecule by ultraviolet or visible light photons is
a phenomenon termed photoluminescence,
which is formally divided into two categories,
fluorescence and phosphorescence
Fluorescence is the property of atoms and
molecules, so called fluorophores, to absorb light
at a particular wavelength and to subsequently
emit light of longer wavelength.
However, Phosphorescence occurs in a manner
similar to fluorescence, but with a much longer
excited state lifetime Figure 1: Aequorea Victoria aka Crystal Jelly [1]
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4. Stokes Shift
British Scientist Sir George G. Stokes who
first described fluorescence in 1852 and
was responsible for coining the term in
honour of the blue-white fluorescent
mineral fluorite (fluorspar)
He discovered the wavelength shift to
longer values in emission spectra that is
known as the Stokes Shift.
However, Plank’s Equation was derived in
1990.
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Figure 2: Fluorophore Absorption and Emission Profiles [2]

5. Jablonski Energy Diagram
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Figure 3: Jablonski Energy Diagram [2]

6. Parameters
Three fundamental parameters commonly used in describing and comparing
fluorophores are as follows:
 Molar Extinction Coefficient (ε): a measure of how strongly a chemical species or
substance absorbs light at a particular wavelength.
 Quantum Yield (Φ): the percentage of photons emitted versus the number
absorbed
 Fluorescence lifetime (τ): the amount of time that a fluorophore spends in the
excited state without emitting a photon
Where the reference wavelength is usually the wavelength of maximum
absorption in the ultraviolet or visible portions of the light spectrum
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7. Glimpse of Fluorescence Microscope
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Figure 4: Epi-Fluorescent Microscope and some output images [3]

8. Filters
 Fluorophores that can absorb a quantity of illumination,
the percentage that will emit secondary fluorescence is
even lower.
 The fundamental problem in fluorescence microscopy is
to produce high-efficiency illumination of the specimen,
while simultaneously capturing weak fluorescence
emission that is effectively separated from the much
more intense illumination band.
 These conditions are satisfied in modern fluorescence
instruments by a combination of filters that coordinate
excitation and emission requirements.
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Figure 5: Filters characteristics [2]

9. Dichromatic Mirror
 It is positioned at 450
angle to both the incident illumination
arriving from the light source, as well as the optical axis of the
microscope
 Light from the source passes through the defined bandpass
excitation filter, and is then reflected down into the objective
by the DM to be focused at the specimen plane
 Fluorescence emission is captured by the objective and
directed back through the DM, which in turn reflects most of
the contaminating excitation light back toward the light source
 Emission wavelengths passing through the DM are further
purified by the emission filter, before traveling to the eyepieces
or the camera image plane
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Figure 6: Dichromatic Mirror[2]

10. Filter and Dichromatic Mirror Characteristics
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Figure 7: Filter and Dichromatic Mirror Characteristics [2]

11. Epi-Fluorescent Microscope
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Figure 8: Epi-Fluorescent Microscope [3]
• Most fluorescence microscopes in
use are Epi-Fluorescence
microscopes, where excitation of
the fluorophore and detection of
the fluorescence are done
through the same light path (i.e.
through the objective).
• These microscopes are widely
used in biology and are the basis
for more advanced microscope
designs

12. Applications of Fluorescence Microscopy
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Figure 9: Application of Fluorescence Microscopy in 1985 [4]

13. Applications of Fluorescence Microscopy
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Figure 10: Application of Fluorescence Microscopy in 2023 [5]

14. Fluorescence Microscopy
ADVANTAGES
 Live Cell Imaging
 High contrast imaging and High specificity
 Quantitative imaging
DISADVANTAGES
 Photobleaching
 Phototoxicity
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15. References
1. https://en.wikipedia.org/wiki/Aequorea_Victoria
2. https://zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html
3. https://www.zeiss.com/microscopy/en/products/light-microscopes/widefield-microscopes/axioscope-5.html
4. Detection, Enumeration, and Sizing of Planktonic Bacteria by Image-Analyzed Epifluorescence Microscopy, MICHAEL E.
et.al., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1985, p. 799-810
5. Pre-hyperglycemia immune cell trafficking underlies subclinical diabetic cataractogenesis, Ranaei Pirmardan et al. Journal of
Biomedical Science (2023) 30:6, https://doi.org/10.1186/s12929-023-00895-6.
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16. Tha
nks
Thanks