Two-dimensional resolution improvement in standing wave microscopy using fast acousto-optic laser scanning

We present a versatile scheme for two-dimensional (2D) resolution enhancement in standing wave fluorescence microscopy (SWFM). This SWFM scheme consists of an interferometer, where both beams are focused at the back focal plane of the objective lens. Their position is controlled by a pair of acousto-optic deflectors (AODs). This results in two collimated beams that interfere in the focal plane, creating a lateral periodic excitation pattern with controlled fringe spacing and orientation. The phase of the standing wave (SW) pattern is controlled by the phase delay between two RF sinusoidal signals driving the AODs. An enlarged fluorescence image formed using the same objective lens is captured by a cooled CCD camera. Data collection involves acquiring images with excitation pattern of three equi-polar orientations (0 degrees, 60 degrees and 120 degrees) and three different phases (0 degrees, 120 degrees, 240 degrees) for each orientation. The SWFM image is algebraically reconstructed from these 9 acquired images. The SWFM image has enhanced 2D lateral resolution of about 100 nm with nearly isotropic effective point-spread function (PSF). As a result of the acousto-optic scanning, the total acquisition time can be as short as 100 mu s and is only further limited by the fluorescence intensity, as well as sensitivity and speed of the CCD camera. Utilizing acousto-optic laser scanning for advanced SWFM provides the exceptional precision and speed necessary for real-time imaging of subresolution processes in living biological systems.