Polarization-controlled TIRFM with focal drift and spatial field intensity correction.
Polarization-controlled TIRFM with focal drift and spatial field intensity correction.
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DOI：
10.1016/j.bpj.2013.12.043
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发表时间：
2014-03
影响因子：
3.4
通讯作者：
Daniel S. Johnson;R. Toledo-Crow;A. Mattheyses;S. Simon
中科院分区：
文献类型：
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作者：
Daniel S. Johnson;R. Toledo-Crow;A. Mattheyses;S. Simon
全内反射荧光显微镜（TIRFM）正在成为一种越来越常见的方法，以缩小照明激发厚度，以研究细胞过程，如胞吐，胞吞和膜动力学。它也经常被用来作为一种方法，以改善信号/噪声在其他技术，如在体外单分子成像，随机光学重建显微镜/光活化定位显微镜成像，和荧光共振能量转移成像。TIRFM独特的照明几何结构还使得能够采用独特的方法来创建用于选择性地激发与光轴平行或垂直对准的荧光团的激发场。这种选择性已被用于研究细胞膜和细胞蛋白质的取向。不幸的是，激光的相干性质，在TIRFM中的典型激发源，经常创建跨照明区域的空间干涉条纹。当对大的细胞区域成像或需要精确定量时，这些条纹特别成问题。已经开发了通过在帧捕获期间调制TIRFM场来最小化这些条纹的方法;然而，这些方法消除了同时激发特定偏振的可能性。一个新的，据我们所知，技术，它补偿空间条纹，同时允许快速图像采集的平行和垂直的激发方向在25毫秒。此外，背反射检测方案的开发，使激发激光器的快速和准确的对准。该探测器还有助于焦点漂移补偿，这是TIRFM中由于窄激发深度而导致的常见问题，特别是在长时间过程中成像或使用灌注流室时。通过使用DiO对活细胞和载玻片上支持的脂质双层（支持的脂质双层）进行膜取向成像来证明该仪器的能力。使用的生物学问题的方法进行了说明，通过检查的时间和空间动态的胞吐囊泡。
Total internal reflection fluorescence microscopy (TIRFM) is becoming an increasingly common methodology to narrow the illumination excitation thickness to study cellular process such as exocytosis, endocytosis, and membrane dynamics. It is also frequently used as a method to improve signal/noise in other techniques such as in vitro single-molecule imaging, stochastic optical reconstruction microscopy/photoactivated localization microscopy imaging, and fluorescence resonance energy transfer imaging. The unique illumination geometry of TIRFM also enables a distinct method to create an excitation field for selectively exciting fluorophores that are aligned either parallel or perpendicular to the optical axis. This selectivity has been used to study orientation of cell membranes and cellular proteins. Unfortunately, the coherent nature of laser light, the typical excitation source in TIRFM, often creates spatial interference fringes across the illuminated area. These fringes are particularly problematic when imaging large cellular areas or when accurate quantification is necessary. Methods have been developed to minimize these fringes by modulating the TIRFM field during a frame capture period; however, these approaches eliminate the possibility to simultaneously excite with a specific polarization. A new, to our knowledge, technique is presented, which compensates for spatial fringes while simultaneously permitting rapid image acquisition of both parallel and perpendicular excitation directions in ∼25 ms. In addition, a back reflection detection scheme was developed that enables quick and accurate alignment of the excitation laser. The detector also facilitates focus drift compensation, a common problem in TIRFM due to the narrow excitation depth, particularly when imaging over long time courses or when using a perfusion flow chamber. The capabilities of this instrument were demonstrated by imaging membrane orientation using DiO on live cells and on lipid bilayers that were supported on a glass slide (supported lipid bilayer). The use of the approach to biological problems was illustrated by examining the temporal and spatial dynamics of exocytic vesicles.