SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking.
SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking.
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DOI：
10.1038/srep44108
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发表时间：
2017-03-13
影响因子：
4.6
通讯作者：
Charbon E
中科院分区：
文献类型：
--
作者：
Antolovic IM;Burri S;Bruschini C;Hoebe RA;Charbon E
在超分辨率定位显微镜中，目前使用sCMOS成像仪（取代EMCCD成像仪）来提高采集速度。单光子雪崩二极管（SPAD）成像仪每比特深度的帧率与sCMOS成像仪相当或更高，同时产生微秒1位帧，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。高时序分辨率还可用于探索荧光染料闪烁和其他光物理性质，可用于染料优化。本文介绍了荧光染料在实验数据上的闪烁分析方法。此外，最近微透镜的使用使SPAD成像仪的整体灵敏度大幅提高（在我们的情况下是12倍），达到了对灵敏度关键应用的满意值。这使我们能够记录使用SPAD成像仪获得的第一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。
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.