Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes
Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes
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DOI：
10.1039/b907837a
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发表时间：
2009-01-01
影响因子：
2.5
通讯作者：
Leake, Mark Christian
中科院分区：
文献类型：
--
作者：
Plank, Michael;Wadhams, George Howard;Leake, Mark Christian
荧光显微镜提供了一种微扰的方法来探测体内生物学。然而，现有的技术是有限的，在灵敏度和时间分辨率为常用的荧光蛋白。在这里，我们提出了一个新的成像系统与诊断工具包，迎合了检测和定量的荧光蛋白用于快速功能成像在单分子水平。它利用定制的显微镜，我们称之为“slimfield”的照明模式，适用于一系列常见荧光蛋白的快速（类似于毫秒）时间分辨率。Slimfield便宜且简单，允许激发强度比宽场成像大100倍，允许高速检测单分子。我们证明了它的应用程序在几个纯化的荧光蛋白作为遗传编码的报告分子的标准使用。受控的体外实验表明，单个蛋白质分子的视野面积约为30 μ m（2），大到足以包裹完整的原核细胞和小的真核细胞。使用一种新的诊断工具包，我们展示了单分子的自动检测和定量，其最大成像速率为128 x 128像素阵列，类似于每秒500帧，这些荧光团的定位精度在50 nm以内。我们报告的第一次在单分子水平的成像的暗淡增强青色荧光蛋白（ECFP）和CyPet。应用修改，我们进行了同步双色薄场成像用于共定位和FRET。我们提出了初步的细菌细胞在体内成像，并证明类似于毫秒级的时间尺度功能成像在单分子水平上可以忽略不计的光损伤。
Fluorescence microscopy offers a minimally perturbative approach to probe biology in vivo. However, available techniques are limited both in sensitivity and temporal resolution for commonly used fluorescent proteins. Here we present a new imaging system with a diagnostic toolkit that caters for the detection and quantification of fluorescent proteins for use in fast functional imaging at the single-molecule level. It utilizes customized microscopy with a mode of illumination we call "slimfield'' suitable for rapid (similar to millisecond) temporal resolution on a range of common fluorescent proteins. Slimfield is cheap and simple, allowing excitation intensities similar to 100 times greater than those of widefield imaging, permitting single-molecule detection at high speed. We demonstrate its application on several purified fluorescent proteins in standard use as genetically-encoded reporter molecules. Controlled in vitro experiments indicate single protein molecules over a field of view of similar to 30 mu m(2) area, large enough to encapsulate complete prokaryotic and small eukaryotic cells. Using a novel diagnostic toolkit we demonstrate automated detection and quantification of single molecules with maximum imaging rates for a 128 x 128 pixel array of similar to 500 frames per second with a localization precision for these photophysically poor fluorophores to within 50 nm. We report for the first time the imaging of the dim enhanced cyan fluoresecent protein (ECFP) and CyPet at the single-molecule level. Applying modifications, we performed simultaneous dual-colour slimfield imaging for use in co-localization and FRET. We present preliminary in vivo imaging on bacterial cells and demonstrate similar to millisecond timescale functional imaging at the single-molecule level with negligible photodamage.