Fluorescent microscopy is a valuable tool in the study
of biological samples. It is a highly sensitive microscopy technique and it can
be used to identify cells and their sub cellular
molecular components. A Fluorescent microscope looks similar to a conventional
light microscope but it relies on very different properties of light to produce
an image. Transmitted light contrasting techniques relies on the following
properties of light to produce an image .
Phase Contrast- phase interference
DIC-Polarization and Phase interference.
Fluorescence microscope is a reflective contrasting technique and it requires
the use of fluorophores or fluorescent proteins to visualize your cells or sub
cellular components. Fluorophores are molecules that when absorbing the energy
of electromagnetic radiation will jump to a higher energy level (excited state).
When some of these molecules return to the ground state they emit radiation.
This is known as fluorescence. Fluorophores have special molecular structures and a
characteristic excitation and emission spectra. Individual fluorophores are
exited within a given wavelength range and will a emit light within a given
wavelength range. The emission wavelength will always be longer than the
Single photon excitation and emission
Excitation and emission spectra of EGFP and Cy5
2-photon fluorescent microscopy involves the use of lower energy light to
excite the sample (higher wavelength IR). For example GFP is normally excited at
around 473nm with one photon excitation. With 2-photon excitation it would be
excited around 843nm.
The advantage is that IR light penetrates deeper into the tissue than shorter
wavelengths. 2- photon excitation only occurs at the focal plane so less
bleaching occurs in the sample above and below the section. Multi Photon
fluorescent microscopy is the technique of choice for deep tissue imaging.
Fluorescence Techniques can be divided into two main
The diagram above is of a basic wide-field inverted fluorescence microscope. The system has
light source( mercury lamp), filter cubes with excitation, emission and dichroic
filters and CCD camera attached.
This is a reflected light method and involved the illumination of the whole
sample. The image will contain in focus light from the image focal plane as well
as out of focus light from above and below the focal plane.
The thicker the sample the more out of focus light will be present in the
image and the more blurred the image will appear. This blurring
caused by the out of focus light can result in loss of resolution and contrast.
Wide-field image of mouse intestine. Out of focus light can cause image blur.
Confocal microscopy is a fluorescence microscopy technique that can be
used to get rid of the out of focus light that causes the problems that we see
in wide-field fluorescence microscopy.
The whole sample is still illuminated as in wide-field but only the light from
the focal plane reached the detector. This is achieved with this use of a small
diaphragm( pinhole) situated in the conjugated focal plane.
Above Image provided by L.Boland Zeiss.co.uk
Confocal image of mouse intestine. Sharp high resolution image no Blur.
Confocal microscopy can be used to reduce the blur in the image and produce
a high contrast fluorescence image. Confocal microscope will allow you to
visualize optical sections of your sample.( ~500nm).
The optical sections can then be reassembled into a high resolution 3D image
FRET ( Fluorescence Resonance Energy Transfer) is another fluorescent
microscopy technique used to investigate molecular interactions. The principal
is bases on the fact that is two fluorescent molecules are close enough together
( A distance or around 1-10nm) then a close acceptor molecule can take the
excitation energy from the donor molecule.
We can measure FRET in different ways:
Acceptor emission-Detect the emission of the acceptor after excitation of the
donor e.g. excite GFP with 488nm but detect RFP at 610nm ( GFP emission at
Donor emission after acceptor bleaching-Take image of donor, then bleach
acceptor (with acceptor excitation wavelength-RFP:580nm), take another image of
donor. This image should be brighter. To image FRET you need to have a suitable
FRET pair ( with overlapping excitation and emmision spectra) The disadvantage
of FRET is that you can see bleed through because of the overlapping spectra.
The fluorescence lifetime is a
measure of how long a photon excited electron will remain in a molecules excited
state before returning to the ground state via the emission of a lower energy
(longer wavelength) photon. When combined with an imaging microscope system it
is possible to measure the fluorescence decay of a molecular species at every
point in the optical field of view. This method is known as Fluorescence
Lifetime Imaging Microscopy (FLIM).The two most commonly used
general techniques to acquire FLIM data are frequency domain and time domain.
More information on
Time domain FLIM
Photoactivation and FRAP:See links below
TIRF see link below: