A fluorescence microscope (colloquially synonymous with

epifluorescent microscope) is a light microscope used to study

properties of organic or inorganic substances using the phenomena of
fluorescence and phosphorescence)

(Olympus BX61), coupled with An inverted

fluorescent microscope
a digital camera
( NikonTE200)
 Uses

Fluorescence microscopy is a rapid expanding technique, both in the

medical and biological sciences.

The technique has made it possible to identify cells and cellular

components with a high degree of specificity.

Certain antibodies and disease conditions or impurities in inorganic

material can be studied with the fluorescence microscopy.
Visible light

Visible light: 400-700 nm

 ‘Fluorescence’ word

‘Fluorescence’ is the luminescence of a substance when it is excited by

radiation. ( chlorophyll and some oils and waxes ).

Fluorescence is the property of some atoms and molecules to absorb light at a

particular wavelength and to subsequently emit light of longer wavelength after a
brief interval.

Specific filters are needed to isolate the excitation and emission wavelengths of a
fluorochrome.

A bright light source with proper wavelengths for excitation is also needed. For
normal fluorescence applications, this is a mercury vapor arc burner.
How does a fluorescence microscope
work?
 Epi-
fluorescence

Suppose..
• The excitation light is violet, and the emitted
light is red. The microscope uses a special
dichroic or dichromatic mirror.
• This mirror reflects light shorter than a certain
wavelength, and passes light longer than that
wavelength. Thus your eye only sees the
emitted red light from the fluorescent dye,
rather than seeing scattered purple light.
• The purple and red bars next to the dichroic
mirror represent additional filters to help
prevent the different wavelengths of light
from going the wrong directions

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Light path in
fluorescent
microscope

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 • A dichroic beam splitter or partial mirror which
reflects lower wavelengths of light and allows higher
wavelengths (lower energy) to pass.

• A beam splitter is required because the objective acts

as a condenser lens for the excitation wavelength as
well as the objective lens for emission.

• This epi-illumination type of light path is required to

create a dark background so that the fluorescence
can be easily seen. The wavelength at which a beam
splitter allows the higher wavelengths to pass must
be set between the excitation and emission
wavelengths of any given fluorochrome so that
excitation light is reflected and emission light is
allowed to pass through it.

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Fluorescence filter setup (Courtesy of Carl Zeiss Inc
Germany).

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Optical filters- examples

Filter
White Light Source Transmitted light

620 -640 nm
Light

Filter
White Light Source Transmitted Light

>520 nm Light

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 Fluorescence microscope
white light -> excitation color ->
specimen
excitation filter

specimen -> emission color -> see it

barrier filter

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Filter cube

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 Specimen preparation

• The fluorescence microscope is based on

the phenomenon that certain material
emits energy detectable as visible light
when irradiated with the light of a specific
wavelength.

• The sample can either be fluorescing in its

natural form like chlorophyll and some
minerals, or treated with fluorescing
chemicals.

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How Sample Gets
Excited?
• First, the microscope has a filter that only lets
through radiation with the desired wavelength that
matches your fluorescing material. The radiation
collides with the atoms in your specimen and
electrons are excited to a higher energy level. When
they relax to a lower level, they emit light.
• To become visible, the emitted light is separated
from the much brighter excitation light in a second
filter.
• 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.

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Absorbs high
energy photon

Emits lower energy photon

Ground state
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 Principle of Fluorescence
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.

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advantages
• Attachment of fluorescent dye molecules to
specific parts of your sample, so that only those
parts are the ones seen in the microscope.

• More than one type of dye can be used.

• By changing the excitation light, you can cause

one type of dye to fluoresce, and then another, to
distinguish two different parts of your sample

• The precise location of intracellular components

labeled with specific fluorophores can be
monitored, as well as their associated diffusion
coefficients, transport characteristics, and
interactions with other biomolecules.
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Light Source

• To generate enough excitation light intensity to furnish

secondary fluorescence emission capable of detection
• Either mercury or xenon arc (burner) lamps is used,
which produce high-intensity illumination powerful
enough to image faintly visible fluorescence specimens.
• Mercury arc burners are very bright lamps with a limited
lifetime and require some maintenance and care to
make sure that they are producing the brightest possible
light beam for fluorescence excitation.

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 CARE AND FEEDING OF A MERCURY ARC
LAMP
• When an arc lamp is brand new and first fired up, it should be
left on for two hours, whether observing specimens or not.

• The lamp should properly aligned to: a) allow for the brightest
fluorescence, and b) to make sure that the real and mirror
images of the arc are not overlapping which may cause
overheating (possible explosion hazard).

• The lamp should always be cold when it is turned on. Never

turn an arc lamp when it is warm. This causes clouding of the
glass in the bulb and both considerably shortens the life of the
burner and decreases the burner's brightness.

• Once a lamp is turned on, it should be left on for a minimum of

twenty minutes (thirty is preferable). This allows it to warm up
properly and will help prevent premature wearing out
(clouding) of the burner.

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 CARE AND FEEDING OF A MERCURY ARC
LAMP
• When an arc lamp is brand new and first fired up, it should be
left on for two hours, whether observing specimens or not.

• The lamp should properly aligned to: a) allow for the brightest
fluorescence, and b) to make sure that the real and mirror
images of the arc are not overlapping which may cause
overheating (possible explosion hazard).

• The lamp should always be cold when it is turned on. Never

turn an arc lamp when it is warm. This causes clouding of the
glass in the bulb and both considerably shortens the life of the
burner and decreases the burner's brightness.

• Once a lamp is turned on, it should be left on for a minimum of

twenty minutes (thirty is preferable). This allows it to warm up
properly and will help prevent premature wearing out
(clouding) of the burner.

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22
Fluorophores
• Many fluorescent probes are constructed
around synthetic aromatic organic chemicals
designed to bind with a biological
macromolecule.
• Fluorescent dyes are also useful in monitoring
cellular integrity (live versus dead and
apoptosis), endocytosis, exocytosis, membrane
fluidity, protein trafficking, signal transduction,
and enzymatic activity.
• Fluorescent probes have been widely applied
to genetic mapping and chromosome analysis
in the field of molecular genetics.
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 Fluorophores

Name Ex. Em.

FITC Blue Green
(Fluorescein)
Alexa-488 Blue Green

eGFP Blue Green

TRITC Green Red

Texas Red Green Red

Propidium Green Red

Iodide (PI)
DAPI violet Blue
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 What is
Photobleaching ?
• Photobleaching is the photochemical destruction of a
fluorophore. In microscopy, photobleaching may complicate
the observation of fluorescent molecules, since they will
eventually be destroyed by the light exposure necessary to
stimulate them into fluorescing.
• Loss of activity caused by photobleaching can be controlled
by reducing the intensity or time-span of light exposure, by
increasing the concentration of fluorophores, or by
employing more robust fluorophores that are less prone to
bleaching (e.g. Alexa Fluors or DyLight Fluors).

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 in a dividing human cancer cell. DNA is stained
blue, a protein called INCENP is green, and the
microtubules are red.
Each fluorophore is imaged separately using a
different combination of excitation and emission
filters, and the images are captured sequentially
using a digital CCD camera, then overlaid to give
a complete image.

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 Multiphoton Fluorescence Microscopy

• Used for imaging living cells and tissues

with three dimensionally resolved
fluorescence imaging.
• Two-photon excitation, which occurs only
at the focal point of the microscope,
minimizes the photobleaching and
photodamage.
• This advantage allows investigations on
thick living tissue specimens that would
not be possible with conventional imaging
techniques.
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 Multiphoton
Fluorescence
Microscopy

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 Advantages of multiphoton imaging:

• Optical sections may be obtained from deeper within

a tissue that can be achieved by confocal or wide-
field imaging. There are three main reasons for this:
The excitation source is not attenuated by absorption
by fluorophore above the plane of focus
Longer excitation wavelengths suffer less scattering
Fluorescence signal is not degraded by scattering
from within the sample as it is not imaged

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 Limitations of multi-photon excitation

• Slightly lower resolution with a given

fluorophore when compared to confocal
imaging. This loss in resolution can be
eliminated by the use of a confocal
aperture at the expense of a loss in signal.
• Thermal damage can occur in a specimen
if it contains chromophores that absorb
the excitation wavelengths, such as the
pigment melanin.
• Only works with fluorescence imaging.
• Currently rather expensive.
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 meiji mx 6000
epi flo micro--
meiji mx meiji tc 5000 epi flo
4050 inverted microscope
epi flo
micro--

VANM GUARD
van guard EPI
advanced – FLUORESCENT
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3032 inverted reaction
3016 EPI FLO. MICROSCOPE microscope
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