Epi-fluorescence microscopy.
Epi-fluorescence microscopy.
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DOI：
10.1007/978-1-62703-056-4_2
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Brown, Claire M
中科院分区：
文献类型：
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作者：
Webb, Donna J;Brown, Claire M
大多数生命科学研究实验室都可以使用落射荧光显微镜，经过优化后可以成为中心实验室工具。本章介绍落射荧光光路并详细讨论各个组件。针对入射灯光源、激发和发射滤光片、二向色镜、物镜和电荷耦合器件 (CCD) 相机提出了建议，以获得最灵敏的落射荧光显微镜。金属卤化物灯的均匀照明与新型“硬”涂层滤光片和镜子、高分辨率单色 CCD 相机以及高 NA 物镜相结合，均推荐用于高分辨率和高灵敏度荧光成像。还建议使用单色相机、电动滤光片转塔、单独的滤光片立方体和相应的染料进行多色成像，这些是敏感、高分辨率多色成像的最佳选择。应使用奈奎斯特采样来收集图像，并应针对背景强度贡献和整个视场的不均匀照明进行校正。吸收大量光（即高消光系数）并产生大量荧光信号（即高量子产率）的光稳定荧光探针和蛋白质是最佳的。还提出了神经元免疫荧光标记方案。最后，为了最大限度地利用灵敏的宽视场显微镜并生成具有高信噪比的最高分辨率图像，提出了将宽视场落射荧光成像与恢复图像反卷积相结合的建议。
Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes that are the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented.