Re-scan confocal microscopy: scanning twice for better resolution

Abstract

Abstract:We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCDcamera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and highsensitivity is required. Toomre, and J. Bewersdorf, "Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms," Nat. Methods 10(7), 653-658 (2013).

Adaptive illumination reduces photobleaching in structured illumination microscopy

“…A number of techniques that outperform standard widefield fluorescence microscope in terms of resolution were developed. These super-resolution methods can be roughly divided in three groups: localization microscopy, which relies on photo-switchable fluorophores and localization of single molecules [1][2][3], structured illumination microscopy, which employs a non-uniform illumination [4][5][6][7][8][9], and stimulated emission depletion microscopy (STED), which uses partial quenching of the fluorophores to narrow down the point spread function [10]. An ideal superresolution fluorescence microscopy technique should allow biologists to observe live cells with improved resolution over extended periods of time.…”

Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples

“…Alternatively, reassignment may be implemented by increasing the distance between adjacent emission foci while leaving their size unchanged (Fig. S1), which provides a convenient method for instant, point-based SIM, as has been demonstrated with single-photon (1P) excitation (“rescan microscopy” [21]). We implemented this same method of reassignment to enhance the resolution of 2P excitation microscopy, using an emission-side galvanometric mirror (galvo) to double the distance between adjacent scan points before image acquisition with a camera (Fig.…”

“…Relative to the recent rescanning microscopy technique [21] (where a detection pinhole is needed to prevent out-of-focus emission from landing on the camera), 2P excitation eliminates the need for a detection pinhole in the emission path because out-of-focus emission does not occur (i.e., optical sectioning is inherent in the excitation process) [Note S1, 27]. This allows direct expansion of the illumination pattern without the need to first descan, thereby simplifying instrument design and alignment.…”

Faster fluorescence microscopy: advances in high speed biological imaging

“…Recent implementations have achieved resolutions of 120 nm laterally and 360 nm axially with volumetric temporal resolution approaching 1 Hz for >100 cellular volumes [35, 36]. Other SIM designs use point-based illumination patterns to confer better sectioning and to improve depth penetration, and have been implemented in point-scanning [37], and parallelized modes [38, 39]. …”