Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution
Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution
复制标题
DOI：
10.1117/1.1805559
复制
发表时间：
2004-11-01
影响因子：
3.5
通讯作者：
Hollywood, MA
中科院分区：
文献类型：
--
作者：
Coates, CG;Denvir, DJ;Hollywood, MA
背照式电子倍增电荷耦合器件 (EMCCD) 相机将尽可能高的光子收集效率与几乎消除读出噪声检测极限的能力相结合，对微光动态细胞显微镜领域产生了深远的影响。我们在这里报告了这款相机的使用情况，采用 512X512 帧传输芯片格式，像素读出速度为 10 MHz，优化了要求严格的超低光细胞内钙通量显微镜设置。所采用的布置包括旋转共焦尼普科夫盘，该盘虽然满足以非常快的帧速率生成图像并最小化背景光子的需要，但产生非常微弱的信号。相机面临的挑战不仅在于检测尽可能多的稀缺光子，还在于以满足许多低光显微镜方法的时间分辨率要求（平滑肌钙流显微镜的特殊要求）的帧速率运行。所提供的结果表明，与之前的超低光 CCD 检测标准（Gen 111+ 增强型电荷耦合器件 (ICCD)）相比，该技术的灵敏度显着提高，并且还展示了 EMCCD 先进的时间和空间分辨率能力。 (C) 2004 年光电仪器工程师协会。
The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512X512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the Gen 111+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD. (C) 2004 Society of Photo-Optical Instrumentation Engineers.