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      LED Zappelin’: An open source LED controller for arbitrary spectrum visual stimulation and optogenetics during 2-photon imaging

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          Two-photon (2P) microscopy is a cornerstone technique in neuroscience research. However, combining 2P imaging with spectrally arbitrary light stimulation can be challenging due to crosstalk between stimulation light and fluorescence detection. To overcome this limitation, we present a simple and low-cost electronic solution based on an ESP32 microcontroller and a TLC5947 LED driver to rapidly time-interleave stimulation and detection epochs during scans. Implemented for less than $100, our design can independently drive up to 24 arbitrary spectrum LEDs to meet user requirements. We demonstrate the utility of our stimulator for colour vision experiments on the in vivo tetrachromatic zebrafish retina and for optogenetic circuit mapping in Drosophila.

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          Independent Optical Excitation of Distinct Neural Populations

          Nathan Klapoetke, Yasunobu Murata, Sung Kim … (2014)
          Optogenetic tools enable the causal examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the examination of how different synapses or pathways interact to support computation. Here we report two new channelrhodopsins, Chronos and Chrimson, obtained through the de novo sequencing and physiological characterization of opsins from over 100 species of algae. Chrimson is 45 nm red-shifted relative to any previous channelrhodopsin, important for scenarios where red light would be preferred; we show minimal visual system mediated behavioral artifact in optogenetically stimulated Drosophila. Chronos has faster kinetics than any previous channelrhodopsin, yet is effectively more light-sensitive. Together, these two reagents enable crosstalk-free two-color activation of neural spiking and downstream synaptic transmission in independent neural populations in mouse brain slice.
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            Optimization of a GCaMP calcium indicator for neural activity imaging.

            Jasper Akerboom, Tsai-Wen Chen, Trevor J. Wardill … (2012)
            Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
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              Deep tissue two-photon microscopy.

              Fritjof Helmchen, Winfried Denk (2005)
              With few exceptions biological tissues strongly scatter light, making high-resolution deep imaging impossible for traditional-including confocal-fluorescence microscopy. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation, providing large depth penetration mainly because even multiply scattered signal photons can be assigned to their origin as the result of localized nonlinear signal generation. Two-photon microscopy thus allows cellular imaging several hundred microns deep in various organs of living animals. Here we review fundamental concepts of nonlinear microscopy and discuss conditions relevant for achieving large imaging depths in intact tissue.
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                Author and article information

                Contributors
                M.J.Y. Zimmermann
                T. Baden
                Journal
                Journal ID (nlm-ta): HardwareX
                Journal ID (iso-abbrev): HardwareX
                Title: HardwareX
                Publisher: Elsevier
                ISSN (Electronic): 2468-0672
                Publication date PMC-release: 22 July 2020
                Publication date Collection: October 2020
                Publication date (Electronic): 22 July 2020
                Volume: 8
                Electronic Location Identifier: e00127
                Affiliations
                [a ]Sussex Neuroscience, School of Life Sciences, University of Sussex, United Kingdom
                [b ]TReND in Africa (www.TReNDinAfrica.org), United Kingdom
                [c ]GOSH Community (http://openhardware.science/)
                [d ]The Francis Crick Institute, London, United Kingdom
                [e ]Institute for Ophthalmic Research, University of Tübingen, Germany
                Author notes
                [* ]Corresponding authors at: Sussex Neuroscience, School of Life Sciences, University of Sussex, United Kingdom (M.J.Y. Zimmermann). m.zimmermann@ 123456sussex.ac.uk t.baden@ 123456sussex.ac.uk
                Article
                Publisher Item ID: S2468-0672(20)30036-5 Publisher ID: e00127
                DOI: 10.1016/j.ohx.2020.e00127
                PMC ID: 9041195
                PubMed ID: 35498254
                SO-VID: 58bfcdb5-8f19-44fb-ae20-1b07fda9af3f
                Copyright © © 2020 The Authors
                License:

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 11 March 2020
                Date revision received : 16 July 2020
                Date accepted : 16 July 2020
                Categories
                Subject: Article

                Keywords: 2 photon,visual neuroscience,optogenetics,light stimulation,led,open hardware
                Data availability:
                Keywords: 2 photon, visual neuroscience, optogenetics, light stimulation, led, open hardware

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