Bansal Vivek, Patel Saumil, Saggau Peter
Baylor College of Medicine, Department of Neuroscience, Houston, Texas 77030, USA.
J Biomed Opt. 2006 May-Jun;11(3):34003. doi: 10.1117/1.2209562.
Due to cellular complexity, studying fast signaling in neurons is often limited by: 1. the number of sites that can be simultaneously probed with conventional tools, such as patch pipettes, and 2. the recording speed of imaging tools, such as confocal or multiphoton microscopy. To overcome these spatiotemporal limitations, we develop an addressable confocal microscope that permits concurrent optical recordings from multiple user-selected sites of interest at high frame rates. Our system utilizes acousto-optic deflectors (AODs) for rapid positioning of a focused laser beam and a digital micromirror device (DMD) for addressable spatial filtering to achieve confocality. A registration algorithm synchronizes the AODs and DMD such that point illumination and point detection are always colocalized in conjugate image planes. The current system has an adjustable spatial resolution of approximately 0.5 to 1 microm. Furthermore, we show that recordings can be made at an aggregate frame rate of approximately 40 kHz. The system is capable of optical sectioning; this property is used to create 3-D reconstructions of fluorescently labeled test specimens and visualize neurons in brain slices. Additionally, we use the system to record intracellular calcium transients at several sites in hippocampal neurons using the fluorescent calcium indicator Oregon Green BAPTA-1.
由于细胞的复杂性，研究神经元中的快速信号传导常常受到以下限制：1. 用传统工具（如膜片吸管）能够同时探测的位点数量；2. 成像工具（如共聚焦或多光子显微镜）的记录速度。为了克服这些时空限制，我们开发了一种可寻址共聚焦显微镜，它能够以高帧率从多个用户选定的感兴趣位点进行同步光学记录。我们的系统利用声光偏转器（AOD）快速定位聚焦激光束，并利用数字微镜器件（DMD）进行可寻址空间滤波以实现共聚焦。一种配准算法使AOD和DMD同步，从而使点照明和点检测始终在共轭像平面中位于同一位置。当前系统的空间分辨率可调，约为0.5至1微米。此外，我们表明记录可以以约40 kHz的总帧率进行。该系统能够进行光学切片；此特性用于创建荧光标记测试标本的三维重建，并可视化脑切片中的神经元。此外，我们使用该系统，利用荧光钙指示剂 Oregon Green BAPTA-1记录海马神经元多个位点的细胞内钙瞬变。
