A total internal reflection fluorescence microscope (TIRFM) is a type of microscope that allows imaging of a thin region of a specimen which can include living cells. 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 cover slip.
The evanescent wave is generated only when the incident light is totally internally 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 to 200 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.
Single-molecule TIRF microscopy has been enabled by technical advances in the fields of optics, materials science, and chemistry, and has been adopted as a standard technique in a number of fields. For example, the breakthrough of high quantum yield fluorophores allowed for high precision tracking of single molecular motors in vitro.
Limitations remain, however, in the number of photons that reach the detector from a single fluorophore, ultimately restricting the temporal resolution of imaging experiments. Efforts to enhance TIRFM have been reported. See, e.g., Kim et al., “Thin-film-based sensitivity enhancement for total internal reflection fluorescence live-cell imaging”, Optics Letters, 2007:32(21):3062-3064 and Kim et al., “Feasibility study of enhanced total internal reflection fluorescence imaging using dielectric films” Multimodal Biomedical Imaging III, Azar et al. Ed., Proc. of SPIE Vol. 6850, 68500R-1 to 68500R-7 (2008). However, a continuing need exists to enhance fluorescence microscopy.