Fluorescence Microscope principle and applications

1. DROSOPHILA CYTOGENETICS LABORATORY
Vinoba Bhave University
Hazaribag
kishore_gupta30@yahoo.com

2. • There is little in the content of most cells
(which are 70% water by weight ) which
impede the passage of light rays.
• Thus most cells in the natural state ,even if
fixed and sectioned ,are almost invisible in
ordinary light microscope
• One way to make them visible under
microscope is to stain them with dyes.

3. • In the early nineteenth centaury non-fluorescence
dyes were used to stain the biological tissues
which showed a preference for particular parts of
the cell like nucleus, mitochondria to make them
visible.
• Today many other specific dyes like malchite
green, sudan black, coommassie blue are used to
reveal sub-cellular component .
• The dye hematoxyline has affinity for negatively
charged molecules so as to reveals the DNA, RNA
and acidic protein in a cell.
• But all these dyes have no specificity at molecular
level

4. Fluorescence Dye
• Use of fluorescence stain/dye has thus increased the
specificity for any protein or other molecules in a cell/tissues
as well as provided high contrast.
For example . Fluorescence when couple to antibody, then in a
cell, where ever it binds with high specificity , can be
detected.
Two types of fluorescence dyes are commonly used
1. Fluorescein- emits green fluorescence when excited with blue
light
2. Rhodamine-emits red fluorescence when green yellow light
3. Many version of these dyes are available commercialy which
with different reactive groups which allow it to bind with
eithe –SH/-NH2 group of protein.
4. Some dyes intercalate for example Hoechest /acridine in
between the double stand DNA

5. Types of Fluorescence Dye
Theses fluorescence molecules absorb light at one wavelength and emit light of
another wave length and then viewed through a filter that allows only light of
emitted wavelength to pass. It is seen to glow against a dark back ground.
Since background id dark even a minute amount of glowing fluorescence dye
can be detected

6. The entire principle of Fluorescence microscopy works on basic concept
Jubsonky’s Diagram –
A diagram representing that the once the
fluorochrome is excited the electron gets
excited and after some time it comes to
ground state and simultaneously energy is
lost in form of light.
Stokes shift - Difference in the
wave length of excited light and
reflected light

7. • Light source -Tungsten and tungsten-halogen
incandescent lamps/laser have been used due
to their emission occurs in the red and infrared
regions of the light spectrum, whereas most
fluorophores are excited by ultraviolet, blue,
and green wavelengths
• Excitation filter - The excitation filter selects a
narrow band of wavelengths from the wide
spectrum generated by the lamp By changing
filter one can select the narrow band as per
desire.
• Emission Filter /Barrier filter – Select the
emitted light of particular color and reject the
other unwanted colored light coming from
back ground . For example barrier filter allow
blue excited light with a wavelength of 450-
490 nm and second barrier filter /emission
filter pass the green fluorescein emission
• Dichoric mirror for transmitting both incident
light and reflected or emitted light

8. Advantage & Applications
• It has great sensitivity. to yield cytochemical information as some normal components
have typical florescence emission. So Vitamin A,. Thiamine, riboflavin and other substances
can be detected.
• The cytochemical value of the method is increased considerably by spectrographic analysis
of the radiation. Some times certain substances incorporated in the cells like
sulphonamides can be localized.
• Lipogenic pigments which are found in the aging person due to increase ages can be
detected. . These lipogenic pigments represent different degree of oxidation &
polymerizations of unsaturated fatty acids.
• With fluorescence dyes like lipofuchsin and ceroid lipogenic pigment bearing cells can be
differentiated.
• Use of para formaldehydes fixative in cryo fixation produces condensation with
catecholamine and
• indolamines which emit green and a yellow fluorescence respectively.
• Used to locate any molecular structure within a cell /tisuues with high sensitivity and
specificity
• can be applied to both live and fixed tissues
• Fate of More than two or three molecules can compared within the same cells.
• Dynamic behaviour of the cells can be revealed

9. •THANKS FOR VISIT

10. •THANKS FOR VISIT