Non-linear evanescent-field imaging

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.