Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation

We propose a new type of total internal reflection fluorescence microscopy (TIRFM) called scanning TIRFM (STIRFM) that uses a focused ring-beam illumination and a high-numerical-aperture objective (NA = 1.65). The evanescent field produced by the STIRFM is focused laterally, producing a small excitation volume that can induce a nonlinear effect such as two-photon absorption. Experimental images of CdSe quantum dot nanocrystals and Rhodamine 6G-doped microbeads show that good lateral and axial resolutions are achieved with the current setup. The theoretical simulation of the focal spot produced in STIRFM geometry shows that the focused evanescent field is split into two peaks because of the depolarization effect of a high numerical-aperture objective lens. However, the point-spread function analysis of both one-photon and two-photon excitation cases shows that the detection of the focus-splitting effect is dependent on the detection pinhole size. The effect of pinhole size on image formation is theoretically investigated and confirmed experimentally with the nanocrystal images.