High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators.
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 microseconds. 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 microns). 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 microns) without any temporal averaging.
Bullen
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Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.
Patel
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SS
Saggau
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D000818:Animals; D002478:Cells, Cultured; D004867:Equipment Design; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology; D007202:Indicators and Reagents; D007477:Ions; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D009930:Organic Chemicals; D012680:Sensitivity and Specificity; D012984:Software; D013997:Time Factors
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007477:Ions; D009930:Organic Chemicals; C071335:calcium orange