Fluorescence ppt

1. Fluorescence Microscopy

2. What is Fluorescence Microscopy?
• Fluorescence - the emission of light after absorption of a
photon.
• Fluorescent molecules - referred to as fluorophores or dyes
– Have distinct absorption, excitation and emission spectra.
• Absorption spectra measure the amount of absorption of
incoming light as a function of wavelength while
• Emission spectra show the intensity of the fluorescence as
a function of wavelength for a constant excitation
wavelength.

3. Cont’d
• A fluorescence microscope is an optical microscope that
uses fluorescence.
• Fluorescence is the emission of light by a substance that
has absorbed light or other electromagnetic radiation.
• Reflection and absorption properties are used to study
organic or inorganic substances.
• Its a method of studying material which can be made to
fluoresce, either in its natural form or when treated with
chemicals capable of fluorescing.

4. Discovery
• British scientist Sir George G. Stokes first described
fluorescence in 1852.
• He observed that the mineral fluorspar emitted red light
when it was illuminated by ultraviolet excitation.
• Stokes noted that fluorescence emission always occurred
at a longer wavelength than of the excitation light.
• This shift towards longer wavelength is known as Stokes
Shift.

5. • The "fluorescence microscope" refers to any microscope
that uses fluorescence to generate an image, whether it
is a more simple set up like an epifluorescence
microscope, or
• A more complicated design such as a confocal microscope,
which uses optical sectioning to get better resolution of
the fluorescent image.

6. Principle
• The specimen is illuminated with light of a specific
wavelength.
• Which is absorbed by the fluorophores, causing them to
emit light of longer wavelengths.
• The illumination light is separated from the much weaker
emitted fluorescence through the use of a spectral
emission filter.

7. • A fluorophore is a fluorescent chemical compound that
can re-emit light upon light excitation.
• Fluorophores typically contain several combined aromatic
groups, or plane or cyclic molecules with several π bonds.
• Typical components of a fluorescence microscope
– Light source
– The excitation filter
– The dichroic mirror and
– The emission filter.
Principle

9. Principle
• A dichroic filter or thin
film filter, is a very
accurate color filter
used to selectively pass
light of a small range of
colors while reflecting
other colors.

10. Principle
• Most fluorescence microscopes in use are epifluorescence
microscopes.
– 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.

11. Principle
• Let excitation light radiate the specimen then sort out
the much weaker emitted light to make up the image.
• The fact that the emitted light is of lower energy and
has a longer wavelength is used.
• The fluorescing areas can be observed in the microscope
and shine out against a dark background with high
contrast

12. Epifluorescence microscopy
• Light of the excitation wavelength is focused on the
specimen through the objective lens.
• The fluorescence emitted by the specimen is focused to
the detector by the objective.
• Since most of the excitation light is transmitted through
the specimen, only reflected excitatory light reaches the
objective together with the emitted light.
• In epifluorescence microscopes, both the illuminated and
emitted light travel through the same objective lens

14. Working of the Fluorescence Microscope
1. Light source- epifluorescence
lamphouse
2. Light of a specific wavelength
(or defined band of wavelengths),
is produced by passing
multispectral light from an arc-
discharge lamp through a
wavelength selective excitation
filter
3. Wavelengths passed by the
excitation filter reflect from the
surface of a dichromatic (also
termed a dichroic) mirror or beam
splitter through the microscope
objective to bathe the specimen
with intense light

15. Working of the Fluorescence Microscope
4. If the specimen fluoresces,
the emission light gathered by
the objective passes back
through the dichromatic mirror
5. It is Filtered by a barrier
(or emission) filter, which
blocks the unwanted excitation

16. Light Source
• Four main types of light source are used, including xenon
arc lamps or mercury-vapor lamps with an excitation
filter, lasers, and high-power LEDs.
• Lasers are mostly used for complex fluorescence
microscopy techniques, while xenon lamps, and mercury
lamps, and LEDs with a dichroic excitation filter are
commonly used for wide field epifluorescence
microscopes.

17. The Dichroic Mirror/dichromatic mirror
•The excitation light reflects off
the surface of the dichroic mirror
into the objective.
•The fluorescence emission passes
through the dichroic to the
eyepiece or detection system.

18. Excitation Filters
• To select the excitation
wavelength, an excitation
filter is placed in the
excitation path just prior
to the dichroic mirror.
Emission Filters
• In order to more specifically
select the emission
wavelength of the light
emitted from the sample and
to remove traces of
excitation light

21. Tonic water Hoechst Fluorescein Rhodamine
Excitation and Emission Spectra

22. Stoke’s Shift
• The emission spectrum of an excited fluorophore is
usually shifted to longer wavelengths when compared to
the absorption or excitation spectrum
– Excitation 495 nm
– Emission: 520 nm
• The intensity of the fluorescence is very weak in
comparison with the excitation light (10-3 to 10-5).
• The emitted light re-radiates spherically in all directions.
• Dark background is required to enhance resolution.

23. • A fluorophore is usually excited at wavelengths near or at
the peak of the excitation curve,
• And detected at widest possible range of emission
wavelengths that include the emission peak
• Absorbs light in the yellow-
green region produces yellow-
orange emission to achieve
maximum fluorescence
intensity

24. Fluorescence microscopy
Fluorochrome Excitation wavelength Emission wavelength
Fluoroscein 490 - blue 520 - green
Rhodamine 550 – green 580 - red
Hoechst (stains DNA) 345 - UV 455 - blue

25. Conventional microscope uses light to illuminate the sample
and produce a magnified image of the sample.
Fluorescence microscope
• Uses a much higher intensity light to illuminate the
sample
• This light excites fluorescence species in the sample,
which then emit light of a longer wavelength.
• A fluorescent microscope also produces a magnified
image of the sample, but the image is based on the
second light source