Nikon TE-2000 & Parikh Lab Capabilities

Optical and fluorescence microscopy is conducted using Nikon’s TE-2000. This system is illuminated with a mercury vapor light source and images captured using objectives ranging from 10-100X. With our current setup, we have the capability to take advantage of a host of techniques including:

FRAP

Fluorescence Recovery After Photobleaching denotes an optical technique capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes. This technique provides great utility in biological studies of cell membrane diffusion and protein binding. In addition, surface deposition of a fluorescing phospholipid bilayer (or monolayer) allows the characterization of hydrophilic (or hydrophobic) surfaces in terms of surface structure and free energy. Similar, though less well known, techniques have been developed to investigate the 3-dimensional diffusion and binding of molecules inside the cell; they are also referred to as FRAP.

TIRF

Total Internal Reflection Microscopy is a characterization technique in which a thin region of a specimen, usually less than 200 nm, can be observed. TIRF was developed by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s. TIRF uses evanescent waves to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass-water interface. Evanescent waves are generated only when the incident light is totally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells as shown in the figure above. The selective visualization of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution. TIRF can also be used to observe the fluorescence of a single molecule, making it an important tool of biophysics and quantitative biology.

FRET

Förster Resonance Energy Transfer describes an energy transfer mechanism between two chromophores. A donor chromophore in its excited state can transfer energy by a nonradiative, long-range dipole-dipole coupling mechanism to an acceptor chromophore in close proximity (typically <10nm). This energy transfer mechanism is termed "Förster resonance energy transfer" (FRET), named after the German scientist Theodor Förster. When both molecules are fluorescent, the term "fluorescence resonance energy transfer" is often used, although the energy is not actually transferred by fluorescence. In order to avoid an erroneous interpretation of the phenomenon that, even when occurring between two fluorescent molecules, is always a nonradiative transfer of energy, the name "Förster resonance energy transfer" may be preferred to "Fluorescence resonance energy transfer".

Please see wikipedia, where these description were taken, for more detail.