title: A new nonscanning confocal microscopy module for functional voltage-sensitive dye and Ca2+ imaging of neuronal circuit activity.
abstract: Recent advances in fluorescent confocal microscopy and voltage-sensitive and Ca(2+) dyes have vastly improved our ability to image neuronal circuits. However, existing confocal systems are not fast enough or too noisy for many live-cell functional imaging studies. Here, we describe and demonstrate the function of a novel, nonscanning confocal microscopy module. The optics, which are designed to fit the standard camera port of the Olympus BX51WI epifluorescent microscope, achieve a high signal-to-noise ratio (SNR) at high temporal resolution, making this configuration ideal for functional imaging of neuronal activities such as the voltage-sensitive dye (VSD) imaging. The optics employ fixed 100- × 100-pinhole arrays at the back focal plane (optical conjugation plane), above the tube lens of a usual upright microscope. The excitation light travels through these pinholes, and the fluorescence signal, emitted from subject, passes through corresponding pinholes before exciting the photodiodes of the imager: a 100- × 100-pixel metal-oxide semiconductor (MOS)-type pixel imager with each pixel corresponding to a single 100- × 100-μm photodiode. This design eliminated the need for a scanning device; therefore, acquisition rate of the imager (maximum rate of 10 kHz) is the only factor limiting acquisition speed. We tested the application of the system for VSD and Ca(2+) imaging of evoked neuronal responses on electrical stimuli in rat hippocampal slices. The results indicate that, at least for these applications, the new microscope maintains a high SNR at image acquisition rates of ≤0.3 ms per frame.
authors.affiliation: Laboratory for Neural Circuit Systems, Institute of Neuroscience, Tokushima Bunri University, Shido, Sanuki, Kagawa, Japan. tominagat@kph.bunri-u.ac.jp
authors.forename: Takashi
authors.initials: T
authors.lastname: Tominaga
authors.forename: Yoko
authors.initials: Y
authors.lastname: Tominaga
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis*; D006624:Hippocampus / Q000737:chemistry; D008297:Male; D018613:Microscopy, Confocal / Q000295:instrumentation*; D051381:Rats; D056969:Voltage-Sensitive Dye Imaging / Q000295:instrumentation*
chemical_list: D002118:Calcium
keywords: Ca2+ imaging; confocal microscopy; hippocampal slice; optical imaging; voltage-sensitive dye