A novel multisite confocal system for rapid Ca2+ imaging from submicron structures in brain slices |
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Authors: | Luiza Filipis Karima Ait Ouares Philippe Moreau Dimitrii Tanese Valeria Zampini Andrea Latini Chun Bleau Charlie Bleau Jeremy Graham Marco Canepari |
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Institution: | 1. Laboratory for Interdisciplinary Physics, UMR 5588 CNRS and Université Grenoble Alpes, Saint Martin d'Hères Cedex, France;2. Laboratories of Excellence, Ion Channel Science and Therapeutics, France;3. Wavefront‐Engineering Microscopy Group, Neurophotonics Laboratory, UMR8250 CNRS and Paris Descartes University, Paris, France;4. CrestOptics Spa, Rome, Italy;5. RedshirtImaging LLC, Decatur;6. SciMeasure Analytical Systems Inc, Decatur, Georgia;7. Cairn Research Ltd, Faversham, UK;8. Institut National de la Santé et Recherche Médicale (INSERM), France |
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Abstract: | In brain slices, resolving fast Ca2+ fluorescence signals from submicron structures is typically achieved using 2‐photon or confocal scanning microscopy, an approach that limits the number of scanned points. The novel multiplexing confocal system presented here overcomes this limitation. This system is based on a fast spinning disk, a multimode diode laser and a novel high‐resolution CMOS camera. The spinning disk, running at 20 000 rpm, has custom‐designed spiral pattern that maximises light collection, while rejecting out‐of‐focus fluorescence to resolve signals from small neuronal compartments. Using a 60× objective, the camera permits acquisitions of tens of thousands of pixels at resolutions of ~250 nm per pixel in the kHz range with 14 bits of digital depth. The system can resolve physiological Ca2+ transients from submicron structures at 20 to 40 μm below the slice surface, using the low‐affinity Ca2+ indicator Oregon Green BAPTA‐5N. In particular, signals at 0.25 to 1.25 kHz were resolved in single trials, or through averages of a few recordings, from dendritic spines and small parent dendrites in cerebellar Purkinje neurons. Thanks to an unprecedented combination of temporal and spatial resolution with relatively simple implementation, it is expected that this system will be widely adopted for multisite monitoring of Ca2+ signals. ![image](//wol-prod-cdn.literatumonline.com/cms/attachment/e82bf941-b2f7-4561-8f25-d639749c4810/jbio201700197-gra-0001.png) |
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Keywords: | brain slices calcium imaging confocal microscopy neurons |
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