High-speed addressable confocal microscopy for functional imaging of cellular activity
High-speed addressable confocal microscopy for functional imaging of cellular activity
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DOI：
10.1117/1.2209562
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发表时间：
2006-05-01
影响因子：
3.5
通讯作者：
Saggau, Peter
中科院分区：
文献类型：
--
作者：
Bansal, Vivek;Patel, Saumil;Saggau, Peter
由于细胞的复杂性，研究神经元中的快速信号通常受到以下限制：1。可以用常规工具（例如贴片移液管）同时探测的位点的数量，以及2.成像工具的记录速度，如共焦或多光子显微镜。为了克服这些时空的限制，我们开发了一种可寻址的共聚焦显微镜，允许并发的光学记录从多个用户选择的网站的兴趣在高帧速率。我们的系统利用声光偏转器（AODs）的快速定位的聚焦激光束和可寻址的空间滤波，以实现共焦的数字微镜器件（DMD）。一种配准算法将AODs和DMD配准，使得点照明和点检测总是共定位在共轭像平面内。目前的系统具有类似于0.5至1 μ m的可调空间分辨率。此外，我们表明，录音可以在聚合帧速率类似于40 kHz。该系统能够进行光学切片;该特性用于创建荧光标记的测试样本的3D重建和可视化脑切片中的神经元。此外，我们使用荧光钙指示剂俄勒冈州绿色BAPTA-1，在海马神经元的几个站点使用该系统记录细胞内钙瞬变。(c)2006年，由光学仪器工程师协会（Society of Photo-Optical Instrumentation Engineers）主办。
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 similar to 0.5 to 1 mu m. Furthermore, we show that recordings can be made at an aggregate frame rate of similar to 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. (c) 2006 Society of Photo-Optical Instrumentation Engineers.