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

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 Ca2+ measurements) and FRAP imaging. Lay description Re-scan Confocal Microscopy (RCM) is a new microscopy technique, 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 camera has high sensitivity and therefore the signal (compared to the noise) in the final image is improved. The ability in distinguishing neighbouring objects in the image, called lateral resolution, of RCM 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 RCM is flexible in its configuration in terms of control of the component of the setup (lasers and filters) and of the movement of the scan and re-scan units. Because of this flexibility, the RCM can be configured to address specific biological applications. In this paper we explore a number of possible configurations of RCM for specific biomedical applications such as multicolour, FRET, ratio-metric (e.g. pH and intracellular Ca2+ measurements) and FRAP imaging.