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首页> 外文会议>Conference on Confocal, Multiphoton, and Nonlinear Microscopic Imaging >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：基于电子乘法CCD的空间分辨荧光相关光谱

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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（FCS）广泛用于对单分子的浓度，扩散系数和动力学的研究。为了在FCS测量中引入空间分辨率，我们开发了一种新型FCS系统，它使用乘法乘以电荷耦合器件（EM-CCD）来在每个像素处获得FCS数据。我们测试了3个样品，其具有不同浓度的荧光珠，并成功地研究了FCS信号的相关系数的差异。此外，我们为基于EM-CCD的FCS测量引入了一种新的照明方法，以限制观察量的深度。尽管蒸发场具有有限的穿透深度的性质，但与横向的分辨率相比，典型的50至200nm的渗透深度短。结果FCS测量在深度方向上变得过于敏感，但在横向方向上变差。因此，我们介绍一种新颖的照明方法，其中激光束入射，角度略小于临界角度，以照亮荧光分子（临界角照明）。观察体积的深度可以通过入射角控制。我们预计该方法可以应用于活细胞中分子局部扩散系数的测量。

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