Evanescent interference patterns for fluorescence microscopy

• Published in: Biophysical journal 1992-02, Vol.61 (2), p.542-552
• Main Authors: Abney, J.R., Scalettar, B.A., Thompson, N.L.
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Biophysical Phenomena; Biophysics; Diffusion; Fluorescence Polarization; Fundamental and applied biological sciences. Psychology; Microscopy, Fluorescence - methods; Photochemistry; Surface Properties
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(92)81858-1

The increasing experimental use of total internal reflection/fluorescence photobleaching recovery has motivated a theoretical study of the spatial intensity profiles generated by two interfering evanescent waves. The interference patterns generated by evanescent waves differ considerably from those generated by plane waves in a homogenous medium because evanescent waves are not transverse and because the evanescent propagation number depends on the incidence angle of the totally internally reflected light. The periodicity and contrast of the evanescent interference patterns under various conditions are calculated; these parameters depend on the intensities, polarizations, and incidence angles of the two incident beams, as well as the refractive indices of the two media that form the planar interface where total internal reflection occurs. The derived intensity profiles are used to develop expressions for the shapes of fluorescence photobleaching recovery curves when evanescent interference patterns are used for fluorescence excitation and bleaching. The calculations also suggest that colliding beam experiments may confirm theoretically predicted evanescent field polarizations.