Super-resolution localization microscopy with large field-of-view for mapping synaptic connectivity at multiple scales (Conference Presentation)

Abstract

Super-resolution localization microscopy (SRLM), including PALM, STORM, dSTORM and many others, achieves ultra-high spatial resolution up to 20~30 nanometers by positioning and reconstructing single molecules from thousands or even tens of thousands of raw images. As intrinsically a wide-field imaging technique, SRLM has the advantage of increasing field-of-view (FOV) without sacrificing either imaging speed or spatial resolution. Currently, limited by the number of active pixels in EMCCD cameras (typically 512 x 512), the maximum FOV of popular SRLM is approximately 50 um x 50 um at the sample plane. Such an FOV is insufficient for observing many biological phenomena which are best interpreted in large FOV, for example, volumetric mapping of synaptic connectivity at multiple scales. In this talk, we will report our recent progresses in the technology development and applications of SRLM with large FOV. We will firstly report the imaging performance of a back-illuminated sCMOS cameras with 95% QE for SRLM. Then, we discuss a high-power homogeneous illumination system which is capable of providing sufficient illumination intensity and excellent illumination homogeneity for SRLM with large FOV. Finally, we present some preliminary results of using large FOV SRLM in mapping synaptic connectivity at multiple scales.