Hamamatsu News 1 / 2018

Re-scan + ORCA-Flash4.0 V3 plug-and-play super resolution confocal microscopy News 2018 Vol. 1 14 Application Report Re-scan Confocal Microscopy The Re-scan Confocal Microscopy (RCM) technology provided by Confocal.nl is a major step towards the accessibility of cutting-edge confocal microscopy for any bioimaging based research institution. The concept, implemented as an add-on module for any existing re­ search grade microscope, allows robust confocal imaging with superior performance in terms of resolution, contrast and light-efficiency. The optical module – simply coupled via C-mount between the microscope and the camera – contains two scanning units for beam­ steering instead of one as in most confocal microscopes. The first scanning unit scans the excitation light across the sample and directs the fluorescence light towards the pinhole, as in any descanned confocal technique. As opposed to standard confocal microscopy, the optical path does not end behind the pinhole with the detector. The beam of fluorescence light is directed to the second scanning unit instead, which projects the image onto a camera. On the one hand, this gives the opportunity to add additional magnification to the system by doubling the speed of the second scanning unit, thereby improving the resolution of the system up to a factor of 2 effectively circumventing Abbe’s diffraction limit without additional computation or sacrificing contrast. Observing fine structures with ideal resolution and contrast On the other hand, the ability to detect confocal images with a modern camera results in additional advantages compared to the standard detection with a photomultiplier tube (PMT). In comparison to PMTs, modern sCMOS cameras show nearly double the quantum efficiency. When comparing this fact with a standard confocal microscope of comparable resolution (with a pinhole of 0.5 Airy Units), the SNR improvement of the RCM technology over conventional confocal microscopy reaches a factor of 4 [1]. Detecting faint signals with the ORCA-Flash4.0 V3 The ORCA-Flash4.0 V3 is a perfect solution for this integral part of the RCM. Of course it provides a very high quantum efficiency of 82 % peak, but images are not defined in terms of quantum efficiency.A more reliable metric of image quality is the image SNR (see box). While the image SNR will always be limited by the shot noise of the available signal, every measurement process will add additional noise to the The image signal is determined by the signal intensity and the quantum efficiency. For the image SNR, the noise sources have to be accounted for. In high signal (or optical background), the noise is mostly composed of shot noise QE · (S+B) and the PRNU. As optical background is a minor issue in confocal techniques like the RCM, minimizing the readout noise σ rn , the dark current shot noise μ I  · t, the DSNU and PRNU maximizes the SNR in all light levels and therefore enables observing the faintest structures. QE · (S+B) + σ rn 2  +μ I  · t exp + (PRNU · QE · (S+B)) 2  +DSNU 2 QE · S Image Signal to Noise QE: Quantum efficiency S: Signal (in photons) B: Background (in photons) σ rn : Readout noise (in electrons rms) μ I : Dark current (in electrons/pixel/second) t exp : Exposure time (in seconds) PRNU: Photo Response Non-Uniformity (in percent) DSNU: Dark Signal Non-uniformity (in electrons)

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