The prism based configuration has been applied to cellular microscopy for nearly 30 years ( Axelrod, 1981). Although the majority of prism based TIRF microscope designs are centered on inverted microscope bases, upright microscopes can also be used (for review, see ( Axelrod, 2001)). One configuration uses a prism to direct laser light toward the interface between the coverglass and media/cells at an incident angle sufficient to cause total internal reflection. There are two common methods for producing the evanescent wave for TIRFM. To my knowledge none of the standard commercially available mounts are suitable for fixed sample TIRFM **Mounting media for fluorescence microscopy can vary substantial. Usually, only fluorophores within 100-200 nm of the coverslip are efficiently excited. The depth of the evanescent wave is a function of the incident illumination angle, wavelength, and refractive index differences ( Equation 2) and its intensity exponentially decays with distance from the coverglass ( Equation 3 Fig 2B). The reflection generates a very thin electromagnetic field (the evanescent wave) on the cell side of the coverslip that has an identical frequency to that of the incident light. At a specific critical angle (θ critical) the beam of light is totally reflected from the glass/water interface, rather than passing through and refracting ( Equation 1 Fig 2A). The refractive index (RI see Table 1 for the refractive indexes of some common reagents) differences between the glass (n 2) and intact cell interior (n 1) regulate how light is refracted or reflected at the interface as a function of incident angle. The basic concept of TIRFM in cellular microscopy is simple: an excitation light beam needs to travel at a high incident angle through a glass coverslip upon which cells are cultured. The following are just a few of the many applications of TIRFM in cellular microscopy. The advantage of such a small illumination volume is three fold: (1) the background is greater than 2,000-fold lower than when imaging by normal epifluorescence microscopy ( Funatsu et al., 1995), which results in a high signal-to-background ratio (2) there is virtually no out-of-focus fluorescence collected (3) cells are exposed to a significantly smaller amount of light ( Fig 1). In comparison, the thickness of a confocal image section is approximately 500 nm. Depending on the excitation wavelength and objective numerical aperture, the thickness of the excitation depth, which is called the evanescent field, can be less than 100 nm from the solid surface. TIRFM is a powerful technique for selectively imaging fluorescent molecules (e.g., GFP, membrane dyes, fluorochromes attached to antibodies, …) in an aqueous environment that are very near a solid substance with a high refractive index (e.g.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |