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SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking

机译:用于超高分辨率定位显微镜的SPAD成像仪可分析快速的荧光团闪烁

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摘要

sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm
机译:当前使用sCMOS成像仪(代替EMCCD成像仪)来提高超分辨率定位显微镜中的采集速度。单光子雪崩二极管(SPAD)成像器的每位深度帧速率与sCMOS成像器相当或更高,同时生成微秒的1位帧而没有读出噪声,从而为深入的时间分辨图像分析铺平了道路。还可以利用高定时分辨率来探索荧光染料闪烁和其他光物理特性,这些特性可用于优化染料。我们在实验数据上介绍了荧光染料的闪烁分析方法。此外,最近使用微透镜已使SPAD成像仪的整体灵敏度大大提高(在我们的情况下为12倍),对于灵敏度要求严格的应用达到了令人满意的值。这使我们能够记录使用SPAD成像仪获得的首个超分辨率定位显微镜结果,定位不确定度为20 nm,分辨率为80 nm

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