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Fluorescence Micros

This document discusses fluorescence microscopy. It explains that fluorescence microscopy uses fluorescence to study organic and inorganic substances, rather than reflection or absorption. It describes the key components of a fluorescence microscope, including the light source, fluorescent dyes, and filters. Finally, it provides examples of applications in microbiology and cell biology, and lists some advantages and limitations of fluorescence microscopy.

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Rishabh ankur
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17 views11 pages

Fluorescence Micros

This document discusses fluorescence microscopy. It explains that fluorescence microscopy uses fluorescence to study organic and inorganic substances, rather than reflection or absorption. It describes the key components of a fluorescence microscope, including the light source, fluorescent dyes, and filters. Finally, it provides examples of applications in microbiology and cell biology, and lists some advantages and limitations of fluorescence microscopy.

Uploaded by

Rishabh ankur
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Cell Biology Fluorescence

Microscopy

Lokender Kumar Ph.D.


Cell biology Lokender Kumar Ph.D.
https://www.biomol.com/dateien/AAT--Fluorescence-Microscopy

Cell biology Lokender Kumar Ph.D.


Fluorescence Microscopy

• Eric Betzig, William Moerner, and Stefan Hell developed super-resolved


fluorescence microscopy and they got Nobel Prize On 8 October 2014.

Cell biology Lokender Kumar Ph.D.


What is Fluorescence Microscopy?
• A fluorescence microscope is a
type of optical microscope that
uses fluorescence instead of,
or in addition to, reflection and
absorption to study properties
of organic or inorganic
substances.

• Fluorescence refers to the Figure 1. Excitation and


emission of light rays from an the shifted emission
profile of Alexa 488.
exciting substance.

Cell biology Lokender Kumar Ph.D.


Parts of Fluorescence Microscope
1.Light Source
• Most of the Fluorescence Microscope use Mercury lamp as a
primary source of light. This emits white lights.

2.Fluorescent dyes or Fluorochromes


• Fluorescent dyes are a type of chemical compounds that emit
light rays when they are excited with UV, blue rays.

Cell biology Lokender Kumar Ph.D.


Parts of Fluorescence Microscope
Excitation filter
• It is a type of filter, which passes the wavelengths absorbed by the
fluorophore.
• Block the excitation light

Emission filter
• It only passes only those wavelengths are emitted from a fluorophore.
• It blocks all unwanted wavelengths

Dichroic mirror
• It is a type of accurate color filter.
• It only passes small range of colors and reflects other colors.
Cell biology Lokender Kumar Ph.D.
Light path…
1. A high-intensity Mercury lamp
2. The exciter filter transmits only blue
lights to the specimen and blocks out all
other colors.
3. The blue light is reflected downward to
the specimen by a dichroic mirror,
which reflects the lights of certain colors
but transmits light of other colors.
4. The specimen is stained with a
fluorescent dye

Cell biology Lokender Kumar Ph.D.


Light path...
5. The stained portion absorbs blue light and
emits green light, which passes upward
penetrates the dichroic mirror and reaches
the barrier filter.
6. This filter allows the green light to pass to the
eye; however, it blocks out any residual blue
lights from the specimen.
7. Thus, the eye perceives the stained portion of
the specimen as glowing green against a black
background whereas the unstained portion of
the specimen is invisible.

Cell biology Lokender Kumar Ph.D.


Application of Fluorescence Microscope
• The Fluorescence microscope has become an essential
tool in medical microbiology

• Bacterial pathogens can be identified after staining


them with fluorescent or specifically labeling them with
fluorescent antibodies using immunofluorescence
produce.

• Another Important use of the fluorescence microscope


is the localization of specific proteins within the cell.

Cell biology Lokender Kumar Ph.D.


Advantages Limitations
• It is used to study the dynamic • During the process of
behavior exhibited in live-cell photobleaching, the Fluorophores
imaging. lose their ability to fluoresce.
• It can trace the location of a • Fluorescent molecules can
specific protein in the cell. generate reactive chemical species
• Due to the presence of higher during the illumination
sensitivity, it can detect the 50 • The specimen must be stained with
molecules per cubic micrometer. the fluorescent dyes.
• It allows multicolor staining of • Specimen preparation is a costly
the specimen. process.

Cell biology Lokender Kumar Ph.D.

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