High speed structured illumination microscopy in optically thick samples

Structured illumination microscopy (SIM) allows imaging of fluorescently labelled biological samples with a spatial resolution improved by a factor of approximately two compared to traditional optical microscopy techniques. The cost of this resolution improvement is the need to capture a number of raw images of the sample to reconstruct a single SIM image, increasing sample light exposure and limiting the ability of the technique to capture dynamic processes. In this paper we describe image acquisition and reconstruction techniques that allow fast super-resolution imaging within optically thick specimens. By exploiting overlaps between SIM information passbands we are able to generate optically sectioned, super-resolution images from an image sequence acquired in a single focal plane. We consider how single plane super-resolution images may be obtained using 2D and 3D SIM illumination patterns, and compare the resulting images to those obtained using conventional 2D SIM reconstruction methods. By combining a single plane reconstruction algorithm with hardware for high-speed switching between illumination patterns and rapid acquisition of fluorescence images, we demonstrate high speed super-resolution imaging inside biological organisms. Crown Copyright (C) 2015 Published by Elsevier Inc. All rights reserved.