Non-linear evanescent-field imaging

Abstract

Total internal reflection fluorescence (TIRF), a general term that embraces any spectroscopic or microscopic technique based on the evanescent field created by TIR of light, is further establishing itself as an important tool for studying near-surface phenomena. Impingement of a femtosecond-pulsed infrared beam on a reflecting interface creates the conditions for ‘macroscopic’ evanescent-field two-photon fluorescence excitation. The two-photon fluorescence excitation volume is confined by both the non-linearity of the multi-photon process and the spatial inhomogeneity of the evanescent field. The absence of scattered excitation resulting in a low background and the possibility of simultaneous multi-colour fluorescence excitation should make non-linear evanescent-field excitation particularly attractive for quantitative single-molecule observation and ultra-sensitive screening assays. In this topical review, we survey the requirements, present the current results and explore the potential of this novel non-linear microscopy.

Total internal reflection spectroscopy for studying soft matter

“…Additionally, the polarisation of the incident light is not preserved at the sample and even the overall light intensity at the sample forms a two lobed shape, and not a simple spot; 23 use of a radially polarised beam returns the shape of the probed volume to a spot 24 but does not allow the S and P polarisations to be independently probed. Objectivebased illumination has a number of appealing features: it does not require significant modification to a standard fluorescence microscope, the objective provides good collection efficiency 25 and the tight focus of annular illumination avoids photobleaching outside the sample volume. 26 However, it also has a number of disadvantages: back-reflected excitation light, the possibility of fluorescence from optical elements in the objective or index-matching fluids, the cost and limited working depth of high NA objectives, sample damage from tightly focussed laser spots, and the limited damage threshold of the objective.…”

“…Two photon fluorescence is a common way to reduce surface scattering in TIRF. 25 In general, scattering from inhomogeneous samples represents a limitation for TIR spectroscopy since it greatly reduces the precision with which the sample volume can be defined.…”

Evanescent Excitation and Emission

“…TIR excitation can be combined with fluorescence recovery after photobleaching (FRAP) or fluorescence correlation spectroscopy (FCS) to measure: (1) diffusion coefficients of surface-proximal fluorophores, in solution, in model or living cell EVANESCENCE IN EXCITATION: TIR membranes, in cell cytoplasm, or even within submicroscopic cellular organelles; 14 (2) chemical kinetic rates of binding/unbinding to the surface; and (3) absolute concentrations of fluorophores (in the case of TIR-FCS). Two-photon TIRF 17 in principle would cut the evanescent characteristic distance d in half. Two-photon TIRF 17 in principle would cut the evanescent characteristic distance d in half.…”

A hydrophilic gel matrix for single-molecule super-resolution microscopy

“…Both methods are well suited for single-molecule localization microscopy due to the increase in signal-to-noise ratio by background reduction. Still, with both methods, experiments might suffer from non-uniform illumination and light scattering, which get more prominent when imaging with multiple channels deep inside the sample (Oheim and Schapper 2005, Rohrbach 2000, van 't Hoff et al, 2008. Multichannel imaging with super-resolution microscopy benefits from homogeneous TIRF and HILO illumination by scanning the focused laser spot in a circular orbit on the back focal plane (van 't Hoff et al, 2008).…”