Fluorescence microscopy uses fluorescence from organic or inorganic dyes to visualize structures within cells or tissues. It provides high resolution and contrast compared to traditional microscopy. The microscope uses a mercury lamp light source and filters to excite fluorescent dyes, which then emit light of longer wavelengths. Common fluorescent dyes include fluorescein and rhodamine. Immunfluorescence microscopy labels structures with fluorescent antibody conjugates. Photobleaching limits fluorescence over time due to excited dye molecules interacting with oxygen before emission.

1. FLUORESCENCE MICROSCOPE
Presented by Subhankar Das roll 104

2. What is Fluorescence microscopy ?
 A fluorescence microscope is an optical microscope that uses
fluorescence and phosphorescence instead of, or in addition to,
reflection and absorption to study properties of organic or
inorganic substances.

3. HISTORY
 In 1845 herschel reported
the first observation of the
fluorescence of a quinine
solution (extracted from
chnchona tree bark) in
sunlight.
 It is a natural crystalline
alkaloid that is sensitive to uv
light and under a black light
it glows blueish.

4. WHY FLUORESCENCS MICROSCOPE ?
 High resolution
 High contrast
 It gives us proper understanding of the different regions inside the
cells.
 This techniques are useful for seeing structures and measuring
physiological and biochemical events in living cells.

5. Fluorescence
 Many substance absorb light of particular wavelength and energy, after
absorbing they emit light of larger wave length called fluorescence.
 The longer the wavelength the lower the energy
 The shorter the wavelength the higher the energy
e.g. UV light from sun causes the sunburn not the red visible light

6. PRINCIPLE
1. Energy is absorbed by the atom
which becomes excited.
2. The electron jumps to a higher
energy level.
3. Soon, the electron drops back to
the ground state, emitting a photon (or
a packet of light) - the atom is
fluorescing

7. PARTS
 Light source: - This is provided by a bright
mercury vapour lamp. This produce light rays in
the range of 200-400 nm and visible rays in the
range of above 780nm.
 Heat Filter:- The rays produced by the lamp
generate considerable heat and certain rays
(infra red) which are of no use in fluorescence.
Heat filter is placed in front of the lamp and
before the condenser to absorb heat. However,
heat filter does not prevent the transmission of
UV and the visible rays.
 Exciter filter:- The light cooled down by the
heat filter next passes through the exciter filter
which absorbs all but shorter waves that are
need to excite the fluorescent dye coated
specimen on the slide. The filters which are
dark allow only green, blue, violet or UV rays.

8. PARTS
 Dichroic Filters: - It is a mirror that reflects one
range of wavelengths and allows another range
to pass through.
 Condenser: For best results, always a dark field
condenser is used because in a dark background
even mild fluorescence can easily be detected.
 Objective lens: - The objective lens collects the
fluorescent-wavelength light produced.
 Barrier filter: - This is situated in the body tube
of the microscope between the objective and the
ocular lens to remove all the remnants of the
exciting light so that only the fluorescence is
seen.
 Ocular lens: - It is the lenses through which
image of the microscopic object is observed.
Depending upon magnification the eye piece is of
four types:- 6x, 10x, 15x, 20x
 CCD camera: One or two ccd camera can be
installed to capture the specimen image.

9. Fluorescence Microscope
upright inverted

10. DICHORIC MIRROR
Dichroic Filters: - It is a mirror that
reflects one range of wavelengths and
allows another range to pass through.

11. DYE
 Most commonly used fluorescent dyes are fluorescein, auramine A, acridine
orange, rhobdamine etc.

12. Stroke Shift
 In1852 Stokes observed that the fluorescing light has longer wavelengths
than the excitation light, a phenomenon that has become to be known as
the Stokes shift.

13. JABLONSKI DIAGRAM
• Jablonski diagram was first
proposed by Professor Alexander
Jablonski in 1935 to describe
absorption and emission of light.
• Upon absorbing a photon of
excitation light, usually of short
wavelengths, electrons may be
raised to a higher energy. (10-15s).
• After going to the higher energy
there is a gap that is referred to as
internal conversion (10-12s).
• Quantum efficiency: is the ratio
between the absorbed and emitted
photon.
• Dye should have a high QE.

14. IMMUNOFLUORESCENCE
 Antibodies to which the fluorescent dye is attached are referred to as labeled antibodies.
 When bacterial cells are incubated with labeled antibodies; the fluorescent dye-antibody conjugate will cover the
surface of the cells.
 The excess fluorescent dye-antibody conjugate is washed off
 Preparation is observed under fluorescence microscope.
 The bacterial cell will glow brilliantly as a result of fluorescence. The cells not covered by the dye do not fluoresce and
hence are not visible by the technique.
 This procedure is known as the fluorescent antibody technique and the phenomenon is termed
immunofluorescence.

15. IMMUNOFLUORESCENCE
 Direct  Indirect

16. PHOTOBLEACHING
 Photobleaching is the irreversible
decomposition of the fluorescent
molecules in the excited state because
of their interaction with molecular
oxygen prior to emission
 Methods for countering photobleaching
• Scan for shorter times
• Use high magnification, high
objective Lens
• Use wide emission filters
• Reduce excitation intensity
• Use “antifade” reagents (not
compatible with viable cells)