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 .

Bright-Field:-Absorption

Dark-Field-Scattering

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 excitation wavelength.

      

Single photon excitation and emission                                                                             Excitation and emission spectra of EGFP and Cy5

2-Photon Microscopy

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 categories:

Standard techniques: 

 wide-field

  confocal

  2-photon

 

Special techniques: 

  FRET

  FLIM

  FRAP

  Photoactivation

  TIRF

The diagram above is of a basic wide-field inverted fluorescence microscope. The system has a polychromatic 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 520nm)

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.

FLIM

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 FLIM Time domain  FLIM frequency domain

 

Photoactivation and FRAP:See links below

Photo-activation and FRAP:

 

TIRF see  link below:

TIRF

 

 

 

 

 

 

 


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Copyright 2009 Beatson Advanced Imaging Reaource
Last modified: 02/25/16