Configurations of the Re‐scan Confocal Microscope (RCM) for biomedical applications

Abstract

The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The signal-to-noise ratio of Re-scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re-scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re-scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re-scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re-scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio-metric (e.g. pH and intracellular Ca measurements) and FRAP imaging.

Imaging cellular ultrastructures using expansion microscopy (U-ExM)

“…For post-U-ExM imaging of clathrin, microutubles and DNA a RCM (Rescanning Confocal Microscope) was used which is based on the image scanning principle where pixel reassignment is purely achieved optomechanically 21. The RCM unit (Confocal.nl) is attached to a sideport of a Nikon TiE and equipped with a sCMOS Zyla 4.2P (Andor).…”

Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

“…For 3D imaging, an additional achromatic cylindrical lens (f = 250 mm, Thorlabs) was placed in the detection path close to the imaging plane before the relay system. Rescanning confocal imaging (RCM) was performed on a Nikon TiE inverted microscope equipped with an RCM unit (Confocal.nl) that is based on the image scanning principle 42 . The setup was operated by the microscope software NIS-Elements (version 4.6).…”

Tracking down the molecular architecture of the synaptonemal complex by expansion microscopy

“…Rescanning confocal microscopy (RCM) imaging was conducted on a Nikon TiE inverted microscope combined with an RCM unit (Confocal.nl). RCM derives from the image-scanning principle, whereby pixel reassignment is carried out purely optomechanically 35,56 . The unit is connected to a Cobolt Skyra (Cobolt, Hübner Group) laser unit providing four excitation laser lines (405, 488, 561, and 640 nm).…”