Super-resolution scanning microscopy with virtually structured illumination

Abstract

The resolution of optical microscopy fundamentally limited by diffraction is at best 200 nm. Super-resolution structured illumination microscopy (SR-SIM) provides an elegant way of overcoming the diffraction limit in conventional widefield microscope by superimposing a grid pattern generated through interference of diffraction orders on the specimen while capturing images. The use of non-uniform illumination field “shift” high specimen frequencies which are out-ofband into the pass-band of the microscope through spatial frequency mixing with the illumination field. Therefore the effective bandwidth of SR-SIM is approximately twice as conventional microscopy, corresponding to a 2-fold resolution enhancement, if the difference between excitation and emission wavelength is ignored. However, such a wide-field scheme typically can only image optically thin samples and is incompatible with multiphoton processes. In this paper, we propose a Super-resolution scanning scheme with virtually structured illumination, utilizes detection sensitivity modulation on line by programming or off line by numerical processing together with temporally cumulative imaging, the excitation intensity is constant while capturing images. In this case a nondescanned array detector such as CCD camera is needed. When combined with multiphoton excitation, this scheme can image thick samples with threedimensional optical sectioning and much improved resolution.