High Speed Resolution Enhancement by Optical Pupil Function Modulating System

Abstract

Superresolution fluorescent microscopy (nanoscopy) offers unprecedented resolution of biological structures by overcoming the fundamental diffraction limit of light. There are several superresolution technologies like stimulated emission depletion (STED), structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM) and fluorescence photoactivation localization (FPALM) all show promise as they offer subdiffraction limit optical resolution. The further challenge in superresolution is the improvements of the temporal resolution, because the temporal resolutions of the above four methods are too low to pursue the movements of proteins at ms timescales. However, it looks so difficult because FPALM and STORM are based on single molecular detection and stochastic photoswitching while STED and SIM require laser illumination scanning. Here, we constructed a microscopic system that can voluntarily modulate optical transmission of the pupil function by placing a reflective liquid crystal micro mirror array (LCMM) onto the Fourier plane that alters the point spread function (PSF) shape. It has been reported that the diffraction limit was overcome by subtracting the normal PSF from the low-pass filtered PSF (∗). Our present system can obtain a subtracted image of superresolve-masked image and the low-pass filtered image within one frame of a camera. We achieved 2 ms temporal resolution at 120 nm spatial resolution without any special probes and scanning systems. Furthermore, because the present technique is compatible with fluorescent probes that lack polarization or coherency, we can apply it to various biological investigations. In this meeting, we show basic principles and constructions of our present nanoscopy, and demonstrate the potentials of it for live cell imaging both on the membrane and intracellular, and for single particle tracking.(∗) Heintzmann R, Sarafis V, Munroe P, Nailon J, Hanley QS, Jovin TM., Micron. 34,293-300 (2003).