Eliminating unwanted far-field excitation in objective-type TIRF. Part I. identifying sources of nonevanescent excitation light.

ABSTRACT: Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using

SUBMITTER: Brunstein M 

PROVIDER: S-EPMC4026778 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature