This presentation shows a basic overview of all aspects of Fluorescence Microscopy including its description, history, mechanism, applications, advantages, limitations, and some examples of studies that used this technique.

1. Fluorescence Microscopy
By: Niharika, Anuj, Devika, Muskan, Harshita, Aayush, and Harsh Raheja
Department of Biochemistry, Shivaji College, University of Delhi

2. INTRODUCTION
What is Fluorescence Microscopy?
Fluorescence is one of the most commonly used physical phenomena in biological and
analytical microscopy, mainly because of its high sensitivity and high specificity.
Fluorescence is a form of luminescence. Fluorescence microscopy even allows users to
determine the distribution of a single molecule species, its amount and its localization inside
a cell.
Fluorescence Microscope
A fluorescence microscope is an optical microscope that uses fluorescence and
phosphorescence instead of, or in addition to, scattering, reflection, and attenuation or
absorption, to study the properties of organic or inorganic substances.

3. Schematic Diagram of Fluorescence
Microscope
● The majority of fluorescence microscopes,
especially those used in the life sciences, are of
the epifluorescence design shown in the
diagram.
● Light of the excitation wavelength illuminates
the specimen through the objective lens.
● The fluorescence emitted by the specimen is
focused to the detector by the same objective
that is used for the excitation.

4. HISTORY
Fluorescence was first discovered in 1845 by Fredrick W. Herschel. He
discovered that UV light can excite a quinine solution (e.g. tonic water) to emit
blue light. British scientist Sir George G. Stokes further studied this discovery,
and he observed that fluorescence emission from an object represents a longer
wavelength than the UV light that originally excited the object.
Many decades later, 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 and
was easily applied to fluorescence microscopy

5. August Kӧhler (1866–1948) working in the Jena Zeiss factory developed in 1893 a new system of microscope
illumination (later named Kӧhler illumination) for microscopic photographic purposes. In 1904, August Kӧhler
invented the ultraviolet absorption microscope that preceded the fluorescence microscope. A camera was required to
detect the very weak image. Kӧhler used the quartz monochromatic ultraviolet objective previously developed by
Moritz von Rohr (1868–1940).

6. mercury-vapor lamp are common; more advanced forms are high-power LEDs and lasers), the
excitation filter, the dichroic mirror (or dichroic beamsplitter), and the emission filter. The filters and
the dichroic beamsplitter are chosen to match the spectral excitation and emission characteristics of
the fluorophore used to label the specimen. In this manner, the distribution of a single fluorophore
(color) is imaged at a time. Multi-color images of several types of fluorophores must be composed by
combining several single-color images. Most fluorescence microscopes in use are epifluorescence
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, such as the confocal microscope and the total internal
reflection fluorescence microscope (TIRF).
TECHNIQUE
The specimen is illuminated with light of a specific
wavelength (or wavelengths) which is absorbed by
the fluorophore, causing them to emit light of
longer wavelengths (i.e., of a 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. Typical components of a fluorescence
microscope are a light source (xenon arc lamp or

7. 1. Quantifying dynamics of maize pollen shed :- Fluorescence microscopy was used to generate digital images of
the trapped pollen (capitalizing on the capacity of pollen to fluoresce) and pollen density per unit area was
counted using commercial imaging software.
2. Investigation of cellular distribution of dinuclear platinum anticancer drugs:- The dinuclear platinum
complexes with aliphatic diamines which are known to be highly active in vitro against several cancer cell lines,
have been modified with a fluorogenic reporter, a hapten (dinitrophenyl, DNP). These labeled complexes have
been designed for fluorescence microscopy investigation of cellular pathways of promising dinuclear platinum
anticancer drugs and present the first example of labeling biologically active dinuclear platinum complexes with a
fluorescent reporter.
3. Non-invasive detection of surface contamination and precursors to laser-induced damage:- Fluorescence
microscopy as a tool to detect surface contamination as well as reveal surface damage precursors on optical
components for large-aperture laser systems, using 351-nm laser excitation.
4. Quantitative access to subcellular dynamics in plant cells:- Fluorescence microscopy to analyse the function of
a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal
resolution in living Arabidopsis cells in their tissue environment.
5. Used in cells as to:-
○ Localisation of specific proteins and other subcellular structures within cells.
○ Identify which cell compartment a protein localises to, and whether it colocalizes with other proteins.
○ Analysis of signalling pathways in individual cells (e.g. calcium imaging).
APPLICATIONS

8. Protein visualised in cells by fluorescence
microscopy
Anticancer drugs working observation using
fluorescence microscopy

9. ADVANTAGES
● It helps to identify the specific molecules of interest by labelling them with the fluorescence
substances
● Also used for visualizing or capturing the standard pattern how the fluorescent substances affect
the cellular structure or tissues at different stages like a heating stage.
● It allows 1-2 magnitude increase in the resolving power and offers a magnified and clear image
of the cellular molecules in the specimen as compared to the traditional optical microscope
● Fluorescence microscopy is the most popular method for studying the dynamic behavior
exhibited in live cell imaging.
● The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer.
● Different molecules can now be stained with different colors, allowing multiple types of 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.

10. LIMITATIONS
● Fluorophore used might interfere with metabolic pathway studied.
Example: GFP is a large protein and might affect movement of tagged protein.
● Excitation light might damage live tissue.
● Excited fluorophore might react with oxygen and generate free radicals toxic to cells.
● Photobleaching: while in excited state fluorophore might undergo covalent
modification that destroys their ability to fluoresce.
● 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 labelled for
fluorescence.
● Largely limited to protein based targets.
● Relatively costly to create new probes.
● Susceptible to autofluorescence.
● Short lifespan of the fluorophore.

11. EXAMPLES
Use of fluorescence microscopy to study intracellular signaling in bacteria -
The quantitative nature and high temporal resolution of fluorescence
microscopy make it particularly useful for studies of intracellular dynamic
systems, such as signaling networks.

12. Thank You