Total internal reflection with fluorescence correlation spectroscopy: Applications to substrate-supported planar membranes

Abstract

In this review paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined.

Total internal reflection spectroscopy for studying soft matter

“…100 The first is to use correlation spectroscopy. Here, only a very small number of molecules (∼100) are in the field-of-view at any one time, 131 by having a small surface coverage, a small sampling area, or by only fluorescently labelling a small fraction of the available adsorbates. The time autocorrelation function of the fluorescence intensity provides information on how long molecules stay within the evanescent wave.…”

“…The time autocorrelation function of the fluorescence intensity provides information on how long molecules stay within the evanescent wave. TIR-fluorescence correlation spectroscopy has been applied widely, for example to dyes at the solid-liquid interface, 132 the interaction of proteins and antibodies, 133 adsorption and binding to supported membranes, 131 , the adsorption and desorption kinetics of different dendrimers 134 and the competitive adsorption of proteins and surfactants. 135 An alternative approach is fluorescence recovery after photobleaching (FRAP or FPR).…”

Total internal reflection fluorescence microscopy imaging-guided confocal single-molecule fluorescence spectroscopy

“…Combining the advantages of spatial resolution of confocal microscopy and total internal reflection microscopy had been well demonstrated by the integrated apparatus in previously reported studies, [17][18][19][20][21][22][23][24][25] which deployed vertical (z-axial) resolution of total internal reflection configuration and lateral resolution of confocal configuration to improve the imaging spatial resolution. In this article, we report our new development of integrated spectroscopy system, which combined the advantages of wide plane view field of objective-type TIRFM imaging with high time-resolution confocal single-molecule spectroscopy to address the challenge of probing both temporally and spatially stochastic events of the single-molecule reactions, such as fluorogenic enzymatic reactions with enzyme tethered to a solution/glass interface.…”

Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy

“…However, in the vicinity of the interface there is a non-propagating evanescent field. This field is used to excite fluorescent molecules in all TIR applications [15]. In objective-based TIR, an excitation field with an irradiance that is approximately Gaussian in the lateral direction, ρ , and exponential in the axial direction, z , is generated [32]: $I(\mathbf{\text{r}})=\eta \frac{2P}{\pi R^2}\text{exp}(-2{\rho }^2/R^2)\text{exp}(-z/d)$…”