Variable-angle Total Internal Reflection Fluorescence Research Articles

Nanotomography of Cell Surfaces with Evanescent Fields

The technique of variable-angle total internal reflection fluorescence microscopy (TIRFM) and its application to nanotomography of cell surfaces are described. Present applications include (1) 3D imaging of chromosomes in their metaphase to demonstrate axial resolution in the nanometre range, (2) measurements of cell-substrate topology, which upon cholesterol depletion shows some loosening of cell-substrate contacts, and (3) measurements of cell topology upon photodynamic therapy (PDT), which demonstrate cell swelling and maintenance of focal contacts. The potential of the method for in vitro diagnostics, but also some requirements and limitations are discussed.

Quantitative analysis of variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) of cell/substrate contacts

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA-TIRFM images are collected from well-spread bovine aortic endothelial cells (BAEC) stained with a membrane-bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64 degrees, both the dorsal and ventral membranes were illuminated, while images excited above 66 degrees illuminated only focal contacts. Above 74 degrees the fluorescence of focal contacts was dominated by back-ground noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 +/- 13 nm. Focal contact areas estimated by TIRFM (1.3 +/- 0.7 micron2) agreed closely with areas observed by immunofluorescence staining of vinculin (1.5 +/- 0.3 microns2).

Depth Profile of Pyrene Dopant in Polymer Films by Variable-Angle Total Internal Reflection Fluorescence Spectroscopy

The incident and observation angular dependences of the fluorescence from pyrene-doped polymer films are measured by variable-angle total internal reflection fluorescence spectroscopy. These angular profiles depend on refractive index, film thickness, observation wavelength, pyrene concentration, absorption coefficient of the polymer film, and so on. The profiles are in good agreement with the results calculated by fitting the parameters and by considering interference effects and concentration profiles. Step profiles of the pyrene concentration explain the results for the segmented poly(urethaneurea) film, while, in poly(methyl methacrylate) and polystyrene films, pyrene is distributed homogeneously.