Two-photon Scanning Fluorescence Microscopy under Total Internal Reflection

Two-photon fluorescence microscopy is a powerful method for many applications. Because of the ability of strong attenuation of the background fluorescence, total internal reflection fluorescence microscopy (TIRFM) has been the major technique in the fluorescence imaging and single molecule detections. Recently, we have developed a new type of TIRFM called scanning TIRFM (henceforth abbreviated STIRFM) which combines the advantages of twophoton fluorescence microscopy and TIRFM [2]. In this system, a high numerical aperture objective is illuminated by a collimated wave and it is thus focused through an interface between a glass cover slip and a sample. An evanescent wave can be produced immediately below the interface due to the total internal reflection caused by the rays of convergence angles larger than the critical angle. The propagating wave produced by the rays of low convergence angles can be suppressed by apodising the objective. As a result, an evanescent focal spot can be formed and an image can be recorded by scanning the sample. This type of STIRFM has higher transverse resolution than conventional TIRFM because of the tight confinement of the evanescent field. Compared with far-field fluorescence microscopy, it has a better suppression of background fluorescence, as its imaging depth is less than 100 nm. The enhancement of the longitudinal polarization at the evanescent focus is also proved useful in fluorescence polarization microscopy. Furthermore, with a highly focused beam, it is ppossible to induce a non-linear effect such as two-photon absorption (see Fig. 1) at the focus. Such STRFM is useful not only in single-molecule detection and three-dimensional polarization microscopy for nanophotonic imaging but also in near-field trapping. Fig. 1 Fluorescence images of CdSe quantum dot nanocrystals on a coverslip glass (10μm μ 10μm) under (a) one-photon (532 nm) and (b) two-photon (800 nm) excitation. Polarization is along the arrow direction. (a) (b) [1] James W. M. Chon and Min Gu, “Scanning total internal reflection fluorescence microscopy and its applications,” Proc. SPIE, 4937 (2002), 202.