Fluorescence Resonance Energy Transfer (FRET) - This technique makes use of two fluorophores, one of which is called the donor and the other of which is called the acceptor. If the emission spectrum of the donor sufficiently overlaps the excitation spectrum of the acceptor, and the transition dipoles of the two fluorophores are oriented close to parallel, and the two fluorophores are between 1 and 10 nm of each other, excitation of the donor will result in excitation of the acceptor by transfer of the excited state energy from donor to acceptor. This is accompanied by a decrease in the emission intensity of the donor compared to the absence of the acceptor. This method has been used to detect intermolecular interactions between two molecules where one is labeled with a donor and one is labeled with an acceptor. Placing the donor and acceptor at different positions on the same molecule has been used to detect conformation changes in proteins. Both these methods have been used to design biosensors for enzyme activity and ligand binding. For further information click here.
Return to top
Two- or multi-photon microscopy (2P or MPM) - This technique depends on the inverse linear relationship between energy and wavelength of light. For example, a photon with a wavelength of 900 nm will have exactly half the energy of a photon with a wavelength of 450 nm. For excitation of a fluorophore, it is the energy, not the wavelength that counts. If two 900 nm photons arrive simultaneously, the fluorophore will be excited and will fluoresce just as if it had absorbed a single 450 nm photon. Similarly, a fluorophore with an excitation maximum of 300 nm can be excited by absorbtion of three 900 nm photons. Simultaneous absorption of more than one photon is a very rare event, but it can be achieved at high photon densities. In 2P or MPM microscopy, a near infrared (NIR) laser is focused on a biological specimen on the stage of a fluorescence microscope. Pumping the laser output to produce femtosecond pulses of very high numbers of photons results in significant two-photon absorbtion by fluorophores in the focal volume. Several advantages of using NIR light recommend this microscopic method for thick, turbid or living biological specimens. Unlike most other wavelengths of light, NIR interacts very little with biological material. This means that outside the focal volume there is little photobleaching, photoxicity, autofluorescence or scattering to degrade the sample or the fluorescence image. In addition, the excitation light is easily filtered out from the emission light. Because there is essentially no excitation of fluorophores outside the focal volume, 2P is inherently confocal with no need for a pinhole aperture to screen out out-of-focus signal. For more information on 2P or MPM click here.
Return to top
Second Harmonic Generation Imaging (SHG) - This technique allows for imaging of endogenous biological structures throughout specimens several hundred micrometers thick without staining or fluorescent labeling. It is analogous to polarization microscopy, in that highly ordered, birefringent structures are detected with high contrast. SHG imaging has been used to visualize collagen, myosin, and tubulin in mitotic spindles in vivo. The SHG signal arises from second-order polarization of ordered arrays of asymmetric molecules, resulting in emission of light at double the frequency (half the wavelength) of the incident light. Thus, NIR laser excitation produces SHG signals in the visible spectrum. SHG can be combined readily with two photon imaging of GFP. For example, upon excitation at 850 nm, GFP fluoresces at the usual emission maximum of 520 nm and any SHG signal can be detected at 425 nm by a relatively simple modification of the microscope. For more information, click here.
Return to top
Fluorescence Correlation Spectroscopy (FCS) - FCS involves the statistical analysis of flucutations in the number and intensity of fluorophores in a microscopic volume of a dilute sample .These fluctuations occur spontaneously as fluorophores enter or leave the sample volume or upon reversible association of two or more fluorescent molecules or with flucutations in the microenvironment of the fluorophore while it is resident in the sample volume. FCS has been most widely used to obtain the diffusion coefficient of fluorescent molecules, but newer methods permit determination of aggregation states and conformational dynamics. The highly focussed laser excitation of the confocal microscope is well suited for FCS in live cells and a commercial attachment (Confocor) is available for the Zeiss LSM 510. For more information, click here.
Return to top
Microcavitation single cell capillary electrophoresis - A pulsed laser beam is used to lyse a single cell. The contents are sucked up into a microcapillary containing agarose or polyacrylamide and the cell contents are then rapidly separated and analysed by electrophoresis. Click here for more information.
Return to top
Total Internal Reflection Fluorescence (TIRF) - A fluorescent sample on a glass coverslip is illuminated obliquely from below by a laser beam or an arc lamp through a high numerical aperture lens so that the angle of incidence of the illumination is greater than the critical angle for total internal reflection. The illuminating light is completely reflected off the coverslip, but an evanescent wave of electromagnetic energy passes into the sample and excites fluorescence. The evanescent wave attenuates with distance into the sample so only fluorophores within about 50 to 100 nm of the coverslip are excited. Instrumentation for this technique is now commercially available from several microscope companies and is being used to study plasma membrane dynamics, including real time movies of exocytosis in neurons and macrophages.
Return to top
Cryoelectron Microscopy - For more information, click here.
Return to top
Positron Emission Tomography (PET) - For more information, click here.
Return to top
(SPECT) - For more information, click here.
Return to top
Magnetic Resonance Imaging (MRI) - For more information, click here.
Return to top
Bioluminescence Imaging - Click here for more information, Bioluminescence Imaging Facility.
Return to top
