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. 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.
On 8 October 2014, the
Nobel Prize in Chemistry
Nobel Prize in Chemistry was awarded to Eric
William Moerner and
Stefan Hell for "the development of
super-resolved fluorescence microscopy," which brings "optical
microscopy into the nanodimension".
1.1 Epifluorescence microscopy
2 Light sources
3 Sample preparation
3.1 Biological fluorescent stains
3.3 Fluorescent proteins
5 Sub-diffraction techniques
Fluorescence micrograph gallery
7 See also
9 External links
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. 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.
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).
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 illuminates 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.
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, highly
uniform illumination with a coefficient of variation of 1-2% can be
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. 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 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.
Main article: 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.
See also: Fluorescent protein
The modern understanding of genetics and the techniques available for
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
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
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
the cells and reveals nothing else about the cell morphologies.
Super resolution microscopy
Super resolution microscopy and Correlative Light-Electron
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
Fluorescence microscopy is central to many techniques
which aim to reach past this limit by specialized optical
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. However, the first experimental demonstration of the 4pi
microscope took place in 1994.
4Pi microscopy maximizes the amount
of available focusing directions by using two opposing objective
Two-photon excitation microscopy
Two-photon excitation microscopy using redshifted light and
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
The first technique to really achieve a sub-diffraction resolution was
STED microscopy, proposed in 1994. This method and all techniques
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). Combining the principle of SPDM with SMI resulted in the
development of the
Vertico SMI microscope. 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).
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
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
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.
Green fluorescent protein
Green fluorescent protein (GFP)
Scanning electron microscope#Cathodoluminescence
Xenon arc lamp
Correlative Light-Electron Microscopy
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Wikimedia Commons has media related to Fluorescent microscope images.
Library resources about
Resources in your library
Resources in other libraries
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)
Quantitative phase-contrast microscopy
Differential interference contrast (DIC)
Second harmonic imaging (SHIM)
Two-photon excitation microscopy
Total internal reflection fluorescence microscopy (TIRF)
Lightsheet microscopy (LSFM/SPIM)
Stimulated emission depletion (STED)
Photo-activated localization microscopy (PALM/STORM)