Fluorescence Microscopy: Principles and Applications
Sir
George
G.
Stokes
e
a
The phenomenon of
fluorescence was known by
the middle of the nineteenth
century. British scientist
Sir
George
G.
Stokes
first
made
the observation
that
the
mineral
fluorspar
exhibits
fluorescence when
illuminated with ultraviolet
light,
and he
coined the
word
"
fluorescence"
Dr.T.V.Rao
MD
1
Differences
between
Conventional
and
Fluorescent
Microscope
e
Dr.T.V.Rao
MD
2
a
The
Conventional
microscope
uses
visible light
(400-700
nanometers)
to
illuminate and
produce a magnified
image of a
sample.
a
A
fluorescence
microscope
,
uses a much
higher
intensity light source
which excites
a
fluorescent species in
a
sample of interest. This
fluorescent species in turn
emits
a
lower energy light
of
a
longer wavelength
that produces the
magnified image instead
of the original light
source.
e
What
is
Fluorescence?
a
Fluorescence
is
light
produced
by a
substance
when it is stimulated
by
another
light.
Fluorescence is called
"cold light"
because
it
does not
come
from
a
hot
source like
an
incandescent light
bulb.
Dr.T.V.Rao
MD
3
a
Fluorescence
microscopy
is a
unique
way of using a
microscope to discover facts about specimens
that
often are not
shown by standard bright field microscopy. In bright field
microscopy, specimens are illuminated from outside,
below
or
above,
and
dark objects are seen
against
a light background. In
fluorescence microscopy, specimens are self-illuminated by
internal light,
so
bright objects are seen in
vivid
color
against
a
dark background. Bright objects
against
dark backgrounds are
more easily
seen.
This characteristic of fluorescence microscopy
makes it very sensitive
and
specific.
Dr.T.V.Rao
MD
4
What
is
Fluorescence
Microscopy?
e
e
Dr.T.V.Rao
MD
5
a
Most cellular components
are
colorless and cannot
be
clearly distinguished under
a
microscope.
The
basic
premise of fluorescence microscopy is to stain the
components with dyes. Fluorescent dyes, also known
as
fluorophores of fluorochromes,
are
molecules that absorb
excitation light
at a
given wavelength (generally UV), and
after
a
short delay
emit
light
at a
longer wavelength.
The
delay between absorption and emission is negligible,
generally on the order of nanoseconds.
The
emission light
can
then
be
filtered from the excitation light to
reveal
the
location of the
fluorophores.
Principle
of
Fluorescent
Microscopy
e
Principle
of
Fluorescent
Microscopy
a
Fluorescence
microscopy
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. The
image
produced is based
on the
second
light source
or the
emission wavelength
of the
fluorescent species -- rather
than
from
the
light originally
used
to
illuminate, and
excite,
the
sample.
Dr.T.V.Rao
MD
6
e
Works
on
Principles
of
Light
Pathways
a
Specifically,
a
dichroic
mirror is
used
to separate
the excitation and
emission light
paths.
Within the objective, the
excitation/emission share
the
same
optics. In
a
fluorescence microscope,
the dichroic mirror
separates the light
paths.
Dr.T.V.Rao
MD
7
e
a
Fluorescence microscopy is the
most
popular method for
studying the dynamic behavior exhibited in
live
cell imaging.
This
stems
from its ability to isolate individual proteins with a
high degree of specificity amidst non-fluorescing
material.
a
The
sensitivity
is
high
enough
to
detect
as
few
as
50
molecules
per
cubic
micrometer.
a
Different molecules can now be stained with different colors,
allowing multiple types of molecule to be tracked
simultaneously. These factors combine to give fluorescence
microscopy a clear advantage over other optical imaging
techniques, for both in vitro
and
in
vivo
imaging.
Dr.T.V.Rao
MD
8
Advantages
of
Fluorescent
Microscopy
Epifluorescence
Microscopy
a
Epifluorescence
microscopy
is
a
e
method
of
fluorescence
microscopy
that
is
widely used
in
life sciences
The excitatory light
is
passed
from
above
(or,
for
inverted
microscopes,
from
below), through the
objective
lens
and then onto the
specimen
instead of passing it first
through
the specimen. The fluorescence
in
the
specimen
gives
rise to
emitted
light which
is focused to
the
detector
by the same
objective
that
is
used
for
the
exc
itation
Dr.T.V.Rao
MD
9
The
Specimens
to
be
Stained
e
a
Most specimens for
fluorescence microscopy
must be
stained.
Fluorescent stains
are
called
"
fluorochromes."
Acridine
orange
,
auramine
O, and
fluorescent antibody
(FA)
are
the fluorochromes
used
most.
Dr.T.V.Rao
MD
10
How
to Use
a
Fluorescence
Microscope
Dr.T.V.Rao
MD
11
How
to
Use
a
Fluorescence
Microscope
a
T
h
e
o
bject
t
o
be
s
t
u
di
ed
i
s
e
marked
with
a
molecule
called
a
fluorophore
(a
dye). When the florescent
light is activated, the light
used
for illumination is
separated from the
florescent molecule (the
fluorophore), which
is
much
weaker.
This
is
done
through
an emission
filter.
Dr.T.V.Rao
MD
12
Step
1
e
a
Lo
ca
t
e
th
e
ligh
t
sw
it
ch
o
n
the side of the
microscope
that turns on the light.
Turn
the microscope
on.
a
Write down the exact time
you turn on the light.
The
florescent light
is
mercury-based, and
a
time log
must be
kept for
exposure and
use
of the
light.
Dr.T.V.Rao
MD
13
e
Step
2
a
Locate
the toggle
switch
on the right
side of the
microscope
between the oculars
and objectives. This
switch
controls the
shutter for the
mercury light to the
objective lens.
Dr.T.V.Rao
MD
14
e
Step
3
a
Select the appropriate dye
for your object (this will
depend entirely on what
you
are
going to
be
studying).
The most
common
dyes include I3
(for
use
with
CTC, DTAF
and fluorescein),
A
(for
use
with
DAPI
and
f420),
N21 (for
use
with Rhoda
mine) and L3 (for
use
Dr.T.V.Rao
MD
with
fluorescein).
20
Step
4
e
a
Put
the
filter
(dye)
into the tray
operated
by
the
silver
sliding knob.
To remove
the
tray,
simply pull the
silver
knob
out.
Dr.T.V.Rao
MD
16
Step
5
e
a
Select the lens you would
like to
use. The 63x
objective lens will have
the highest numerical
aperture.
The 100x
objective lens will have
the highest magnitude
that
can be used
with the
mercury-based
florescent
light
source.
Dr.T.V.Rao
MD
17
Step
6
e
a
Turn
the light off when
finished,
and
mark the time.
Wait
30
minutes before
turning the light back on, or
the lamp could explode. It is
a good idea to keep track of
how
many
hours the lamp
is
in use
and
replace it
according to the
manufacture's guidelines.
Dr.T.V.Rao
MD
18
Step
7
e
a
Clean
off the
microscope lens
with lens paper,
or
if really
dirty,
use
a
cotton
swap
and
glass
cleaner.
Dr.T.V.Rao
MD
19
Fluorescence microscopy, pioneered by British scientist Sir George G. Stokes, reveals hidden details in specimens using fluorescent dyes that emit light of longer wavelengths. This innovative technique allows for visualization of cellular components that are otherwise colorless under conventional microscopy. By exciting fluorophores with high-intensity light, fluorescence microscopy provides sensitive and specific imaging, distinguishing bright objects on dark backgrounds. Explore the fascinating world of fluorescence microscopy to uncover intricate details about specimens.
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Presentation Transcript
Sir George G. Stokes e a The phenomenon of fluorescence was known by the middle of the nineteenth century. British scientist Sir George G. Stokes firstmade the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence" Dr.T.V.RaoMD 1
Differences between Conventional and Fluorescent Microscope e aThe Conventional microscope uses visible light (400-700 nanometers) to illuminate and produce a magnified image of a sample. a A fluorescence microscope, uses a much higher intensity light source which excites a fluorescent species in a sample of interest. This fluorescent species in turn emits a lower energy light of a longer wavelength that produces the magnified image instead of the original light source. Dr.T.V.RaoMD 2
What is Fluorescence? e aFluorescence islight produced by a substance when it is stimulated by another light. Fluorescence is called "cold light" because it does not come from a hot source like an incandescent light bulb. Dr.T.V.RaoMD 3
What is Fluorescence Microscopy? e a Fluorescence microscopy is a unique way of using a microscope to discover facts about specimens that often are not shown by standard bright field microscopy. In bright field microscopy, specimens are illuminated from outside, below or above, and dark objects are seen against a light background. In fluorescence microscopy, specimens are self-illuminated by internal light, so bright objects are seen in vivid color against a dark background. Bright objects against dark backgrounds are more easily seen. This characteristic of fluorescence microscopy makes it very sensitive and specific. Dr.T.V.RaoMD 4
Principle of Fluorescent Microscopy e a Most cellular components are colorless and cannot be clearly distinguished under a microscope. The basic premise of fluorescence microscopy is to stain the components with dyes. Fluorescent dyes, also known as fluorophores of fluorochromes, are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength. The delay between absorption and emission is negligible, generally on the order of nanoseconds. The emission light can then be filtered from the excitation light to reveal the location of the fluorophores. Dr.T.V.RaoMD 5
Principle of Fluorescent Microscopy e Fluorescence microscopy 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. The image produced is based on the second light source or the emission wavelength of the fluorescent species -- rather than from the light originally used to illuminate, and excite, the sample. a Dr.T.V.RaoMD 6
Works on Principles of Light Pathways e a Specifically, a dichroic mirror is used to separate the excitation and emission light paths. Within the objective, the excitation/emission share the same optics. In a fluorescence microscope, the dichroic mirror separates the light paths. Dr.T.V.RaoMD 7
Advantages of Fluorescent Microscopy e a Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live cell imaging. This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-fluorescing material. a The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer. a Different molecules can now be stained with different colors, allowing multiple types of molecule to be tracked simultaneously. These factors combine to give fluorescence microscopy a clear advantage over other optical imaging techniques, for both in vitro and in vivo imaging. Dr.T.V.RaoMD 8
Epifluorescence Microscopy Epifluorescence microscopy is a e method of fluorescence microscopy that is widely used in life sciences The excitatory light is passed from above (or, for inverted microscopes, from below), through the objective lens and then onto the specimen instead of passing it first through the specimen. The fluorescence in the specimen gives rise to emitted light which is focused to the detector by the same objective that is used for the excitation a Dr.T.V.RaoMD 9
The Specimens to be Stained e a Most specimens for fluorescence microscopy must be stained. Fluorescent stains are called "fluorochromes." Acridine orange, auramine O, and fluorescent antibody (FA) are the fluorochromes used most. Dr.T.V.RaoMD 10
How to Use a Fluorescence Microscope Dr.T.V.RaoMD 11
How to Use a Fluorescence Microscope a The object to be studied ise marked with a molecule called a fluorophore (a dye). When the florescent light is activated, the light used for illumination is separated from the florescent molecule (the fluorophore), which is much weaker. This is done through an emission filter. Dr.T.V.RaoMD 12
Step 1 e a Locate the light switch on the side of the microscope that turns on the light. Turn the microscope on. a Write down the exact time you turn on the light. The florescent light is mercury-based, and a time log must be kept for exposure and use of the light. Dr.T.V.RaoMD 13
Step 2 e aLocate the toggle switch on the right side of the microscope between the oculars and objectives. This switch controls the shutter for the mercury light to the objective lens. Dr.T.V.RaoMD 14
Step 3 e a Select the appropriate dye for your object (this will depend entirely on what you are going to be studying). The most common dyes include I3 (for use with CTC, DTAF and fluorescein), A (for use with DAPI and f420), N21 (for use with Rhoda mine) and L3 (for use Dr.T.V.Rao MDwith fluorescein). 20
Step 4 e aPut the filter (dye) into the tray operated by the silver sliding knob. To remove the tray, simply pull the silver knob out. Dr.T.V.RaoMD 16
Step 5 e a Select the lens you would like to use. The 63x objective lens will have the highest numerical aperture. The 100x objective lens will have the highest magnitude that can be used with the mercury-based florescent light source. Dr.T.V.RaoMD 17
Step 6 e a Turn the light off when finished, and mark the time. Wait 30 minutes before turning the light back on, or the lamp could explode. It is a good idea to keep track of how many hours the lamp is in use and replace it according to the manufacture's guidelines. Dr.T.V.RaoMD 18
Step 7 e aClean off the microscope lens with lens paper, or if really dirty, use a cotton swap and glass cleaner. Dr.T.V.RaoMD 19
