Hamamatsu News 1 / 2018

News 2018 Vol. 1 15 Cos7 cells imaged with the RCM. Nucleus is displayed in blue, microtubuli in grays and mitochondria in violet. The width of the volume is 45 μm. The confocal stack was rendered using ClearVolume [3]. shot noise, reducing the final image SNR. This additional noise is composed of the readout noise of the detector, as well as the dark current shot noise. The additional noise limits the SNR in the regime where the signal is very low and the the quantum efficiency limits the SNR in high signal conditions. While the dark current shot noise can be neglected in many situations when the exposure time is short, confocal techniques like RCM require exposure times in the range of 1s to 4s. The dark current shot noise is defined as μ I t exp with μ I being the dark current and t exp being the exposure time, meaning the dark current shot noise scales with t exp . The ORCA-Flash4.0 V3 has a very low readout noise of 0.8 e - median (1.4 e - rms), but also an exceptional low dark current of 0.06 e - /px/s (0.006 e - /px/s at maximum cooling), resulting in an additional dark current shot noise with a 1s exposure of only 0.2 e - (0.1 e - at maxi­ mum cooling). To avoid any further non-uniformity in the image, which might limit the information content of the experiment, each camera is individually calibrated to minimize the dark signal non-uniformity and the photo response non-uniformity inherent in CMOS image sensors [2]. The combination of the RCM with the ORCA-Flash4.0 V3 is an ideal all-around solution, whether you are interested in a high performance system to observe faint structures with good SNR up to a resolution of 170 nm, or simply want an affordable, easy-to-use plug-and-play confocal system. [1] De Luca, Giulia, et al. “Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity.” Methods and applications in fluorescence 5.1 (2017): 015002. [2] European Machine Vision Association. “EMVA Standard 1288, Standard for Characterization of Image Sensors and Cameras.” Release 3 (2010): 29. [3] Royer, Loic A., et al. “ClearVolume: open-source live 3D visualization for light-sheet microscopy.” Nature methods 12.6 (2015): 480.

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