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Fluorescnec 1

Fluorescence microscopy allows visualization of structures and processes in cells and tissues by using fluorescent labels. It works by exciting fluorochrome molecules with specific wavelengths of light, causing them to emit light of longer wavelengths. Key events included Herschel discovering fluorescence in 1845, Stokes describing it in fluorite in 1842, and Jablonski developing the diagram explaining fluorescence in 1935. Major applications include immunofluorescence, tracking intracellular molecules and interactions, and examining cellular functions like endocytosis.

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37 views

Fluorescnec 1

Fluorescence microscopy allows visualization of structures and processes in cells and tissues by using fluorescent labels. It works by exciting fluorochrome molecules with specific wavelengths of light, causing them to emit light of longer wavelengths. Key events included Herschel discovering fluorescence in 1845, Stokes describing it in fluorite in 1842, and Jablonski developing the diagram explaining fluorescence in 1935. Major applications include immunofluorescence, tracking intracellular molecules and interactions, and examining cellular functions like endocytosis.

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Fluorescence Microscopy

 Luminescence

1. Fluorescence 2. Phosphorescence

 Fluorochromes and Fluorophores

 Excitation and Emission - Jablonski diagram

 Stokes shift

 Fluorescence microscope

 Applications
Fluorescence Microscopy

 In 1845, Sir John Frederick William Herschel


(1792-1871) discovered the phenomenon of
fluorescence in a quinine solution.
Quinine

Herschel

 In 1842, Sir George Gabriel Stokes (1819-1903)


described the luminescence observed in the
fluorspar mineral (calcium fluoride) as fluorescence.

George Gabriel Stokes


 In 1911, Max Haitinger (1868-1946), who is
considered the founder of modern fluorescence
microscopy and the fluorochroming technique,
coined the term “fluorochrome”.
Max Haitinger

 In 1935, Alexander Jablonski (1898-1980)


presented his diagram to explain fluorescence: a
fluorochrome features different energy states, the
so-called singlets (S0, S1, S2...).

 Beginning in 1950, Albert Hewett Coons (1912-


1978) and Melvin Kaplan developed the
Immunofluorescence technique that led to
sensational results in cell research, an area of natural
sciences.
Albert Hewett Coons
Fluorescence Microscopy
 Phenomena of fluorescence:

Emission of light from any substance is called “Luminescence “ and occurs


from electronically excited states

 Luminescence is divided into two categories (depending on the nature of


the excited state)
1. Fluorescence 2. Phosphorescence

 Fluorescence is the emission of light from excited singlet state

 Phosphorescence is the emission of light from triplet excited state

 Typical fluorescence life time is 10 ns

 Phosphorescence life time is milliseconds to seconds

 Fluorescence typically occurs from aromatic substances 131


Fluorescence Microscopy
 Some chemical substances absorbs light of a particular wavelength and
energy, and emit light of longer wavelength within nanoseconds.

 Such substances are called fluorescent and phenomenon is termed


fluorescence

 When fluorescent materials are illuminated by short wave (UV light), they
give off visible light, glowing brightly against a dark background

 The difference between the exciting


(absorbing) and emitted wavelengths
known as stokes shift.

 This property, makes fluorescence so


powerful
This principle is applied in Fluorescence
Microscopy
 Useful to detect fluorescent molecules 133
Excitation / emission
Jablonski diagram
Jablonski diagram, first proposed by Professor
Alexander Jablonski in 1935
 Some microorganisms are naturally fluorescent. Ex: photosynthetic
bacteria and algae

 But most microorganisms are non fluorescent

 Fluororoscent dyes are used to make them fluorescent

 Fluorescent dyes are called fluorochromes

 DAPI, Hoechst dye – used to label nuclear DNA

 Rhodamine labelled phalloidine – used to label cytoplasmic actin filaments

 Flurochrome labelled Ab - Immunofluorescence

 New tagging methods developed

Epitope tagging - Insertion of short DNA

Constructing protein chimeras with green and red fluorescent protein 136
Hoechst 33342

DAPI

Rhodamine

Epitope tagging
Normalized absorption and fluorescence emission spectra
of fluorescein-conjugated IgG
Principles of operation of a fluorescence microscope

 Light source
 Heat filter
 Exciter filter
 Mirror
 Dark field condenser
 Specimen
 Objective lens
 Barrier filter
 Eyepiece

141
How does Fluorescent Microscopy Work?
Principles of operation of a fluorescence microscope
 Mercury vapor arc lamp or other source produces an intense beam of light

 Special infrared filter limits the heat transfer

 Exciter filter transmits only the desired wavelength of light.

 Darkfield condenser provides a black background against which the


fluorescent objects glow.

 Specimens are stained with dye molecules, called fluorochromes

 Specimens fluoresce brightly upon exposure to light of a specific


wavelength

 Some microorganisms are autofluorescing.

 Barrier filter removes any remaining ultraviolet light, which could damage
the viewer’s eyes, or blue and violet light, which would reduce the image’s
contrast.

 Image of the fluorochrome-labeled specimen forms from the light emitted


from specimen (fluorescence).
Fluorescence Upright Biological Microscope
(a) E. Coli with fluorescent antibodies (b) Paramecium with acridine orange

(c) Flagellate protozoa (d) Mixture of Micrococcus and Bacillus

145
Fluorescence microscopy of endothelial cells using three labels. Red lables
the mitochondria, green lables the F-actine cytoskeleton and blue lables
the nucleus.
Fluorescent micrograph of Ashbya gossypii

Fluorescent micrograph of Ashbya


gossypii mycelium with DAPI labeled
nuclei
Blue: nuclei (DNA), red: outline of the cell

Riboflavin
Applications of fluorescence microscopy
 Useful to detect fluorescent molecules

 The amount , intracellular location and movement of macromolecules,


small metabolites and ions can be studied

 Molecular mechanism of large variety of macromolecules and


metabolites

 Binding dissociation process

 Interaction between proteins using FET (fluorescence energy transfer)

 Study of cell viability and apoptosis

 Examination of cell function such as endocytosis, exocytosis, signal


transduction

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