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首页> 外文会议>Confocal, Multiphoton, and Nonlinear Microscopic Imaging III; Progress in Biomedical Optics and Imaging; vol.8 no.43; Proceedings of SPIE-The International Society for Optical Engineering; vol.6630 >Spatially resolved fluorescence correlation spectroscopy based on electron multiplying CCD
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Spatially resolved fluorescence correlation spectroscopy based on electron multiplying CCD

机译：基于电子倍增CCD的空间分辨荧光相关光谱

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摘要

Fluorescence correlation spectroscopy (FCS) is widely used for investigation of concentration, diffusion coefficients and dynamics of single molecules. To introduce spatial resolution in FCS measurement, we develop a novel FCS system, which uses an electron-multiplying charge-coupled device (EM-CCD) to get FCS data at each pixel. We tested 3 samples, which have different concentrations of fluorescent beads, and successfully investigated the difference of correlation coefficients of FCS signal. In addition, we introduce a new illumination method for EM-CCD based FCS measurement, to limit depth of a observation volume. Although a evanescent field has a nature of limited penetration depth, the penetration depth which is 50 to 200nm in typical, is short in comparison with the resolution in the lateral direction. As a result FCS measurement becomes too sensitive in the depth direction, but worse in lateral direction. So we introduce a novel illumination method, in which a laser beam is incident with an angle slightly smaller than the critical angle to illuminate fluorescent molecule (critical-angle illumination). The depth of observation volume can be controlled with the angle of incidence. We expect this method to be applied to a measurement of local diffusion coefficient of molecules in living cells.
机译：荧光相关光谱法（FCS）被广泛用于研究单分子的浓度，扩散系数和动力学。为了在FCS测量中引入空间分辨率，我们开发了一种新颖的FCS系统，该系统使用电子倍增电荷耦合器件（EM-CCD）来获取每个像素处的FCS数据。我们测试了3个样品，它们具有不同浓度的荧光珠，并成功地研究了FCS信号相关系数的差异。另外，我们引入了一种新的照明方法，用于基于EM-CCD的FCS测量，以限制观察体积的深度。尽管a逝场的穿透深度有限，但与横向分辨率相比，典型的穿透深度通常为50至200nm。结果，FCS测量在深度方向上变得过于敏感，而在横向方向上变得更差。因此，我们介绍了一种新颖的照明方法，其中以比临界角稍小的角度入射的激光束来照明荧光分子（临界角照明）。观察体积的深度可以用入射角控制。我们希望该方法可用于测量活细胞中分子的局部扩散系数。

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