Total internal reflection fluorescence

Silica Colloidal Crystals as Porous Substrates for Total Internal Reflection Fluorescence Microscopy of Live Cells

“…Finally, total internal reflection microscopy allows the use of polarization, which enables study of the orientation of bound ligands. 9 It is presently not known to what extent the cell membrane bends to follow the contour of the colloidal crystal, and bending of the membrane would give an artificially broadened orientational distribution. Smaller colloids, or colloids with patterned surface coverages, could resolve these issues.…”

“…6 In artificial bilayers, TIRF affords surface selectivity to distinguish bound from unbound ligands, 7,8 and TIRF enables the control of polarization to provide orientational information. 9 Very high index substrates allow depth profiling on the scale of tens of nanometers by changing the penetration depth of the evanescent wave. 10 The use of cell membrane fragments allows for a native environment while still avoiding the complications of live cells, such as receptor internalization and autofluorescence.…”

Total Internal Reflection Fluorescence Microscopy: Applications in Biophysics

“…However, in practice, inverse Laplace transforms can yield results that are artifactual. Consequently, this method requires careful attention to a number of factors including the degree to which the accuracy of the data influences the accuracy of the inverse transform, the use when possible of reference samples, the possibility that the fluorophores or the medium in which they are embedded alter the local refractive index, the special emission properties of fluorophores next to interfaces (see below), possible deleterious effects arising from scattered evanescent illumination, and instrumental factors such as optical convolution in the x-y plane and image acquisition time [8,[85][86][87][88][89][90][91][92][93].Alternative approaches to inverse Laplace transforming are to specify an approximate model for the theoretical form of C(z) [85] or to examine the ratio of fluorescence intensities measured at two different incidence angles [94,95], although these approaches do not necessarily circumvent all of the potential complicating factors listed above. Higher refractive index substrates are useful in VA-TIRF because they have a lower critical angle for internal reflection, and therefore enable a wider range of incidence angles and evanescent wave depths [89,90].…”

“…The emission properties of fluorescent molecules next to planar dielectric interfaces, modeled as classical oscillating electric dipoles, are dramatically altered from the properties in homogeneous space.A long history of theoretical work has predicted that the nearby interface will cause significant changes in the angular dependence of the emitted fluorescence, the fluorescence lifetime, and the total collected fluorescence given a particular optical geometry [8,86,[122][123][124][125][126][127]. These surface-induced changes in fluorescence emission depend on a variety of factors including the emission dipole orientation relative to the interface, the distance from the surface to the dipole, the quantum efficiency of the fluorophore in homogeneous space, and the optical details of fluorescence collection.…”

“…The use of polarized evanescent excitation is thus particularly applicable to interfacial samples because, in most of these samples, the orientational anisotropy averaged over an optically accessible area is found primarily in the polar rather than the azimuthal angle relative to the normal to the interface. The conceptual and theoretical basis of using P-TIRFM for characterizing transition dipole orientation distributions at surfaces has been extensively developed, including descriptions of measurements not only of the linear absorption dichroism, but also the polarization of the emitted fluorescence [67,86,91,125,126,129,130]. The use of P-TIRFM to examine the orientation distribution of fluorophores at surfaces has a variety of applications.…”