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Snapgene confocal microscopy1/4/2023 ![]() A multi-spot version of the rescan concept has been proposed by Shroff and York. The rescan concept is very recently disclosed as well by Heintzmann and associates who subsequently published a theory paper on the rescan concept during the review process of the current paper. The improvement of lateral resolution in RCM was quantified by imaging of fluorescent beads of 100 nm, showing that the FWHM is reduced down to 170 nm ( ± 10 nm). In contrast with Structured Illumination techniques, RCM is an “optics only” super resolution technique, meaning that no image reconstruction procedure is required to get sharp images. In addition to improved resolution, RCM has superior light collection efficiency due to a relatively open pinhole and use of a sensitive EMCCD camera as detector. This de-coupling of magnification is the fundamental concept of RCM that gives lateral super-resolution with conservation of optical sectioning. In Re-scan Confocal Microscopy (RCM) we use a separate pair of re-scanning mirrors for a flexible optical architecture which allows de-coupling of the scanning magnification of the object and the magnification of the scanning spot. In the nineties, the concept of re-scanning was also implemented in microscopes with a stationary slit aperture for fast confocal imaging. RCM is based on standard confocal microscopy, extended with an optical unit (re-scanner) that projects, in a scanning way, the image directly on a camera. Here, we propose a new fully optical technique, Re-scan Confocal Microscopy (RCM), that overcomes the limitations on acquisition and processing time of ISM, while maintaining the advantages in (lateral and axial) resolution improvement. ISM allows imaging with good sectioning properties and improved lateral resolution, but the acquisition time is still a significant drawback. Throughout the years, several groups worked on the implementation of ISM for both scanning with a single focus and scanning with multiple foci at the same time. ![]() Subsequently, an image reconstruction algorithm calculates the final image with enhanced lateral resolution a time consuming image acquisition and processing procedure. ![]() In this technique, named Image Scanning Microscopy (ISM) in ref, the camera takes an image of the distribution of emitted light for every position of the excitation focus in the sample. More than 20 years ago Sheppard proposed to solve this problem by removing the pinhole and using a camera instead at the original position of the pinhole. Therefore, in practice most microscopists adjust the pinhole radius to one Airy unit (equal to 1.2λ/NA) thus sacrificing lateral resolution for the sake of signal-to-noise ratio. This, however, would reduce the detection efficiency dramatically and result in images with a very low signal-to-noise ratio. The reason why is clear: lateral resolution can only be reduced by minimizing the pinhole diameter (in theory down to zero). Remarkably, the historical success of the confocal microscope was not caused by its improved lateral resolution but by its sectioning properties (axial resolution). Due to the stormy developments in the field of super-resolution microscopy, it is often forgotten that confocal microscopy was the first super-resolution technique giving improvement of lateral resolution by a factor of √2 compared to wide-field fluorescence microscopy. ![]()
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