High-speed, random-access fluorescence microscopy .1. High-resolution optical recording with voltage-sensitive dyes and ion indicators

被引:99
作者
Bullen, A
Patel, SS
Saggau, P
机构
[1] BAYLOR COLL MED,DIV NEUROSCI,HOUSTON,TX 77030
[2] UNIV PENN,SCH MED,DEPT PHYSIOL,PHILADELPHIA,PA 19104
关键词
D O I
10.1016/S0006-3495(97)78086-X
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
The design and implementation of a high-speed, random-access, laser-scanning fluorescence microscope configured to record fast physiological signals from small neuronal structures with high spatiotemporal resolution is presented. The laser-scanning capability of this nonimaging microscope is provided by two orthogonal acousto-optic deflectors under computer control. Each scanning point can be randomly accessed and has a positioning time of 3-5 mu s. Sampling time is also computer-controlled and can be varied to maximize the signal-to-noise ratio. Acquisition rates up to 200k samples/s at 16-bit digitizing resolution are possible. The spatial resolution of this instrument is determined by the minimal spot size at the level of the preparation (i.e., 2-7 mu m). Scanning points are selected interactively from a reference image collected with differential interference contrast optics and a video camera. Frame rates up to 5 kHz are easily attainable. Intrinsic variations in laser light intensity and scanning spot brightness are overcome by an on-line signal-processing scheme, Representative records obtained with this instrument by using voltage-sensitive dyes and calcium indicators demonstrate the ability to make fast, high-fidelity measurements of membrane potential and intracellular calcium at high spatial resolution (2 mu m) without any temporal averaging.
引用
收藏
页码:477 / 491
页数:15
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