Question

Describe the techniques of total internal reflection fluorescence (TIRF) microscopy, and give an example of its application in life science. Most TIRF in vivo studies investigate membrane complexes; why is this? Can TIRF be applied to monitoring the nucleus of cells?

Answer 1

Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices. In practice, the most commonly utilized interface in the application of TIRFM is the contact area between a specimen and a glass coverslip or tissue culture container.The availability of complete ready-to-use instrumentation systems for employment of the method, as well as developments in fluorophore technology, such as genetically encoded fluorescent species, have made it possible to investigate a number of cell membrane and other surface processes in a direct manner that was not previously possible.

Principal on which it works: Total Internal Reflection

Basic Instrumental Approaches to TIRFM:There are two basic approaches to configuring an instrument for total internal reflection fluorescence microscopy: the prism method, and the objective lens method.

TIRFM Applications: Total internal reflection illumination has potential benefits in any application requiring imaging of minute structures or single molecules in specimens having large numbers of fluorophores located outside of the optical plane of interest, such as molecules in solution in Brownian motion, vesicles undergoing endocytosis or exocytosis, or single protein trafficking in cells. Such specimens typically exhibit a dramatic increase in signal-to-noise ratio from restriction of the excitation region thickness.TIRFM is an ideal tool for investigation of both the mechanisms and dynamics of many of the proteins involved in cell-cell interactions.

Most TIRF in vivo studies investigate membrane complexes: Live-cell imaging represents one of the most promising applications of the TIRFM technique. Protein interactions at the cell membrane surface, such as those involved in focal adhesions, have tremendous importance in cell biology. An understanding of the signals involved in normal cell growth and its attenuation resulting from cell-cell contacts (contact inhibition) may provide insight into abnormal cell growth that occurs in diseases such as cancer.

TIRF be applied to monitoring the nucleus of cells: No, it can not be used because total internal reflection fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching effect by lasers in TIRF microscopy is considerably less when using fluorescent nanoparticles and it reduces photo-induced cytotoxicity during visualization of live-cell events since it only illuminates the specific area near or at the plasma membrane.