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Fluorescence

microscope

An upright fluorescence microscope (Olympus BX61)


with the fluorescent filter cube turret above the
objective lenses, coupled with a digital camera.
Play media
Fluorescent and confocal microscopes operating
principle

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.[1][2] 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.[3]

On 8 October 2014, the Nobel Prize in


Chemistry was awarded to Eric Betzig,
William Moerner and Stefan Hell for "the
development of super-resolved
fluorescence microscopy," which brings
"optical microscopy into the
nanodimension".[4][5]
Principle
The specimen is illuminated with light of a
specific wavelength (or wavelengths)
which is absorbed by the fluorophores,
causing them to emit light of longer
wavelengths (i.e., of a different color than
the absorbed light). The illumination light
is separated from the much weaker
emitted fluorescence through the use of a
spectral emission filter. Typical
components of a fluorescence microscope
are a light source (xenon arc lamp or
mercury-vapor lamp are common; more
advanced forms are high-power LEDs and
lasers), the excitation filter, the dichroic
mirror (or dichroic beamsplitter), and the
emission filter (see figure below). The
filters and the dichroic beamsplitter are
chosen to match the spectral excitation
and emission characteristics of the
fluorophore used to label the specimen.[1]
In this manner, the distribution of a single
fluorophore (color) is imaged at a time.
Multi-color images of several types of
fluorophores must be composed by
combining several single-color images.[1]

Most fluorescence microscopes in use are


epifluorescence microscopes, where
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, such as
the confocal microscope and the total
internal reflection fluorescence
microscope (TIRF).

Epifluorescence microscopy

Schematic of a fluorescence microscope.


The majority of fluorescence microscopes,
especially those used in the life sciences,
are of the epifluorescence design shown in
the diagram. 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 same
objective that is used for the excitation
which for greater resolution will need
objective lens with higher numerical
aperture. Since most of the excitation light
is transmitted through the specimen, only
reflected excitatory light reaches the
objective together with the emitted light
and the epifluorescence method therefore
gives a high signal-to-noise ratio. The
dichroic beamsplitter acts as a wavelength
specific filter, transmitting fluoresced light
through to the eyepiece or detector, but
reflecting any remaining excitation light
back towards the source.

Light sources
Fluorescence microscopy requires intense,
near-monochromatic, illumination which
some widespread light sources, like
halogen lamps cannot provide. Four main
types of light source are used, including
xenon arc lamps or mercury-vapor lamps
with an excitation filter, lasers,
supercontinuum sources, and high-power
LEDs. Lasers are most widely used for
more complex fluorescence microscopy
techniques like confocal microscopy and
total internal reflection fluorescence
microscopy while xenon lamps, and
mercury lamps, and LEDs with a dichroic
excitation filter are commonly used for
widefield epifluorescence microscopes. By
placing two microlens arrays into the
illumination path of a widefield
epifluorescence microscope,[6] highly
uniform illumination with a coefficient of
variation of 1-2% can be achieved.
Sample preparation

A sample of herring sperm stained with SYBR green in


a cuvette illuminated by blue light in an
epifluorescence microscope. The SYBR green in the
sample binds to the herring sperm DNA and, once
bound, fluoresces giving off green light when
illuminated by blue light.

In order for a sample to be suitable for


fluorescence microscopy it must be
fluorescent. There are several methods of
creating a fluorescent sample; the main
techniques are labelling with fluorescent
stains or, in the case of biological samples,
expression of a fluorescent protein.
Alternatively the intrinsic fluorescence of a
sample (i.e., autofluorescence) can be
used.[1] In the life sciences fluorescence
microscopy is a powerful tool which
allows the specific and sensitive staining
of a specimen in order to detect the
distribution of proteins or other molecules
of interest. As a result, there is a diverse
range of techniques for fluorescent
staining of biological samples.
Biological fluorescent stains

Many fluorescent stains have been


designed for a range of biological
molecules. Some of these are small
molecules which are intrinsically
fluorescent and bind a biological molecule
of interest. Major examples of these are
nucleic acid stains like DAPI and Hoechst
(excited by UV wavelength light) and
DRAQ5 and DRAQ7 (optimally excited by
red light) which all bind the minor groove
of DNA, thus labeling the nuclei of cells.
Others are drugs or toxins which bind
specific cellular structures and have been
derivatised with a fluorescent reporter. A
major example of this class of fluorescent
stain is phalloidin which is used to stain
actin fibres in mammalian cells.

There are many fluorescent molecules


called fluorophores or fluorochromes such
as fluorescein, Alexa Fluors or DyLight
488, which can be chemically linked to a
different molecule which binds the target
of interest within the sample.

Immunofluorescence

Immunofluorescence is a technique which


uses the highly specific binding of an
antibody to its antigen in order to label
specific proteins or other molecules within
the cell. A sample is treated with a primary
antibody specific for the molecule of
interest. A fluorophore can be directly
conjugated to the primary antibody.
Alternatively a secondary antibody,
conjugated to a fluorophore, which binds
specifically to the first antibody can be
used. For example, a primary antibody
raised in a mouse which recognises
tubulin combined with a secondary anti-
mouse antibody derivatised with a
fluorophore could be used to label
microtubules in a cell.

Fluorescent proteins
The modern understanding of genetics
and the techniques available for modifying
DNA allow scientists to genetically modify
proteins to also carry a fluorescent protein
reporter. In biological samples this allows
a scientist to directly make a protein of
interest fluorescent. The protein location
can then be directly tracked, including in
live cells.

Limitations
Fluorophores lose their ability to fluoresce
as they are illuminated in a process called
photobleaching. Photobleaching occurs as
the fluorescent molecules accumulate
chemical damage from the electrons
excited during fluorescence.
Photobleaching can severely limit the time
over which a sample can be observed by
fluorescent microscopy. Several
techniques exist to reduce photobleaching
such as the use of more robust
fluorophores, by minimizing illumination,
or by using photoprotective scavenger
chemicals.

Fluorescence microscopy with fluorescent


reporter proteins has enabled analysis of
live cells by fluorescence microscopy,
however cells are susceptible to
phototoxicity, particularly with short
wavelength light. Furthermore, fluorescent
molecules have a tendency to generate
reactive chemical species when under
illumination which enhances the
phototoxic effect.

Unlike transmitted and reflected light


microscopy techniques fluorescence
microscopy only allows observation of the
specific structures which have been
labeled for fluorescence. For example,
observing a tissue sample prepared with a
fluorescent DNA stain by fluorescent
microscopy only reveals the organization
of the DNA within the cells and reveals
nothing else about the cell morphologies.
Sub-diffraction techniques
The wave nature of light limits the size of
the spot to which light can be focused due
to the diffraction limit. This limitation was
described in the 19th century by Ernst
Abbe and limits an optical microscope's
resolution to approximately half of the
wavelength of the light used. Fluorescence
microscopy is central to many techniques
which aim to reach past this limit by
specialized optical configurations.

Several improvements in microscopy


techniques have been invented in the 20th
century and have resulted in increased
resolution and contrast to some extent.
However they did not overcome the
diffraction limit. In 1978 first theoretical
ideas have been developed to break this
barrier by using a 4Pi microscope as a
confocal laser scanning fluorescence
microscope where the light is focused
ideally from all sides to a common focus
which is used to scan the object by 'point-
by-point' excitation combined with 'point-
by-point' detection.[7] However, the first
experimental demonstration of the 4pi
microscope took place in 1994.[8] 4Pi
microscopy maximizes the amount of
available focusing directions by using two
opposing objective lenses or Two-photon
excitation microscopy using redshifted
light and multi-photon excitation.

Integrated correlative microscopy


combines a fluorescence microscope with
an electron microscope. This allows one to
visualize ultrastructure and contextual
information with the electron microscope
while using the data from the fluorescence
microscope as a labelling tool.[9]

The first technique to really achieve a sub-


diffraction resolution was STED
microscopy, proposed in 1994. This
method and all techniques following the
RESOLFT concept rely on a strong non-
linear interaction between light and
fluorescing molecules. The molecules are
driven strongly between distinguishable
molecular states at each specific location,
so that finally light can be emitted at only a
small fraction of space, hence an
increased resolution.

As well in the 1990s another super


resolution microscopy method based on
wide field microscopy has been
developed. Substantially improved size
resolution of cellular nanostructures
stained with a fluorescent marker was
achieved by development of SPDM
localization microscopy and the structured
laser illumination (spatially modulated
illumination, SMI).[10] Combining the
principle of SPDM with SMI resulted in the
development of the Vertico SMI
microscope.[11][12] Single molecule
detection of normal blinking fluorescent
dyes like Green fluorescent protein (GFP)
can be achieved by using a further
development of SPDM the so-called
SPDMphymod technology which makes it
possible to detect and count two different
fluorescent molecule types at the
molecular level (this technology is referred
to as two-color localization microscopy or
2CLM).[13]
Alternatively, the advent of photoactivated
localization microscopy could achieve
similar results by relying on blinking or
switching of single molecules, where the
fraction of fluorescing molecules is very
small at each time. This stochastic
response of molecules on the applied light
corresponds also to a highly nonlinear
interaction, leading to subdiffraction
resolution.

Fluorescence micrograph
gallery
A z-projection of an osteosarcoma cell
phalloidin stained to visualise actin
filaments. The image was taken on a
confocal microscope and the subsequent
deconvolution was done using an
experimentally derived point spread
function.
Epifluorescent imaging of the three
components 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.
Endothelial cells under the microscope.
Nuclei are stained blue with DAPI,
microtubules are marked green by an
antibody bound to FITC and actin
filaments are labeled red with phalloidin
bound to TRITC. Bovine pulmonary artery
endothelial (BPAE) cells
3D dual-color super-resolution microscopy
with Her2 and Her3 in breast cells,
standard dyes: Alexa 488, Alexa 568.
LIMON microscopy
Human lymphocyte nucleus stained with
DAPI with chromosome 13 (green) and 21
(red) centromere probes hybridized
(Fluorescent in situ hybridization (FISH))
Yeast cell membrane visualized by some
membrane proteins fused with RFP and
GFP fluorescent markers. Imposition of
light from both of markers results in yellow
color.
Super-resolution microscopy: Single YFP
molecule detection in a human cancer cell.
Typical distance measurements in the
15 nm range measured with a Vertico-
SMI/SPDMphymod microscope
Super-resolution microscopy: Co-
localization microscopy (2CLM) with GFP
and RFP fusion proteins (nucleus of a
bone cancer cell) 120.000 localized
molecules in a wide-field area (470 µm2)
measured with a Vertico-
SMI/SPDMphymod microscope
Fluorescence microscopy of DNA
Expression in the Human Wild-Type and
P239S Mutant Palladin.
Fluorescence microscopy images of sun
flares pathology in a blood cell showing
the affected areas in red.

See also
Green fluorescent protein (GFP)
Mercury-vapor lamp
Microscope
Scanning electron
microscope#Cathodoluminescence
Stokes shift
Xenon arc lamp
Correlative Light-Electron Microscopy

References
1. Spring KR, Davidson MW. "Introduction to
Fluorescence Microscopy" . Nikon
MicroscopyU. Retrieved 2008-09-28.
2. "The Fluorescence Microscope" .
Microscopes—Help Scientists Explore
Hidden Worlds. The Nobel Foundation.
Retrieved 2008-09-28.
3. Juan Carlos Stockert, Alfonso Blázquez-
Castro (2017). Fluorescence Microscopy in
Life Sciences . Bentham Science
Publishers. ISBN 978-1-68108-519-7.
Retrieved 17 December 2017.
4. Ritter, Karl; Rising, Malin (8 October
2014). "2 Americans, 1 German win
chemistry Nobel" . Associated Press.
Retrieved 8 October 2014.
5. Chang, Kenneth (8 October 2014). "2
Americans and a German Are Awarded
Nobel Prize in Chemistry" . New York
Times. Retrieved 8 October 2014.
6. F.A.W. Coumans; E. van der Pol; L.W.M.M.
Terstappen (2012). "Flat-top illumination
profile in an epi-fluorescence microscope by
dual micro lens arrays" . Cytometry Part A.
81 (4): 324–331.
doi:10.1002/cyto.a.22029 .
PMID 22392641 .
7. Cremer, C; Cremer, T (1978).
"Considerations on a laser-scanning-
microscope with high resolution and depth
of field" (PDF). Microscopica acta. 81 (1):
31–44. PMID 713859 .
8. S.W. Hell, E.H.K. Stelzer, S. Lindek, C.
Cremer; Stelzer; Lindek; Cremer (1994).
"Confocal microscopy with an increased
detection aperture: type-B 4Pi confocal
microscopy". Optics Letters. 19 (3): 222–
224. Bibcode:1994OptL...19..222H .
doi:10.1364/OL.19.000222 .
PMID 19829598 .
9. Baarle, Kaitlin van. "Correlative
microscopy: Opening up worlds of
information with fluorescence" . Retrieved
2017-02-16.
10. Hausmann, Michael; Schneider,
Bernhard; Bradl, Joachim; Cremer, Christoph
G. (1997), "High-precision distance
microscopy of 3D nanostructures by a
spatially modulated excitation fluorescence
microscope", in Bigio, Irving J;
Schneckenburger, Herbert; Slavik, Jan; et al.,
Optical Biopsies and Microscopic
Techniques II (PDF), Optical Biopsies and
Microscopic Techniques II, 3197, p. 217,
doi:10.1117/12.297969
11. Reymann, J; Baddeley, D; Gunkel, M;
Lemmer, P; Stadter, W; Jegou, T; Rippe, K;
Cremer, C; Birk, U (2008). "High-precision
structural analysis of subnuclear complexes
in fixed and live cells via spatially
modulated illumination (SMI) microscopy"
(PDF). Chromosome research : an
international journal on the molecular,
supramolecular and evolutionary aspects of
chromosome biology. 16 (3): 367–82.
doi:10.1007/s10577-008-1238-2 .
PMID 18461478 .
12. Baddeley, D; Batram, C; Weiland, Y;
Cremer, C; Birk, UJ (2003). "Nanostructure
analysis using spatially modulated
illumination microscopy" (PDF). Nature
Protocols. 2 (10): 2640–6.
doi:10.1038/nprot.2007.399 .
PMID 17948007 .
13. Gunkel, M; Erdel, F; Rippe, K; Lemmer, P;
Kaufmann, R; Hörmann, C; Amberger, R;
Cremer, C (2009). "Dual color localization
microscopy of cellular nanostructures"
(PDF). Biotechnology journal. 4 (6): 927–38.
doi:10.1002/biot.200900005 .
PMID 19548231 .

External links
Wikimedia Commons has media related
to Fluorescent microscope images.

Fluorophores.org , the database of


fluorescent dyes
Microscopy Resource Center
Fluorescence Microscopy at Leica
Science Lab
animations and explanations on various
types of microscopes including
fluorescent and confocal microscopes
(Université Paris Sud)

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