An official website of the United States government
Two-photon calcium imaging provides large-scale recordings of neuronal activities at cellular resolution. A robust, automated and high-speed pipeline to simultaneously segment the spatial footprints of neurons and extract their temporal activity traces while decontaminating them from background, noise and overlapping neurons is highly desirable to analyse calcium imaging data. Here we demonstrate DeepCaImX, an end-to-end deep learning method based on an iterative shrinkage-thresholding algorithm and a long short-term memory neural network to achieve the above goals altogether at a very high speed and without any manually tuned hyperparameter. DeepCaImX is a multi-task, multi-class and multi-label segmentation method composed of a compressed sensing-inspired neural network with a recurrent layer and fully connected layers. The neural network can simultaneously generate accurate neuronal footprints and extract clean neuronal activity traces from calcium imaging data. We trained the neural network with simulated datasets and benchmarked it against existing state-of-the-art methods with in vivo experimental data. DeepCaImX outperforms existing methods in the quality of segmentation and temporal trace extraction as well as processing speed. DeepCaImX is highly scalable and will benefit the analysis of mesoscale calcium imaging.
more »
« less
Bright and photostable chemigenetic indicators for extended in vivo voltage imaging
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.
more »
« less
- Award ID(s):
- 1707398
- PAR ID:
- 10174259
- Author(s) / Creator(s):
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Date Published:
- Journal Name:
- Science
- Volume:
- 365
- Issue:
- 6454
- ISSN:
- 0036-8075
- Page Range / eLocation ID:
- 699 to 704
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila. These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.more » « less
-
Abstract Accurately mapping changes in cellular membrane potential across large groups of neurons is crucial for understanding the organization and maintenance of neural circuits. Measuring cellular voltage changes by optical means allows greater spatial resolution than traditional electrophysiology methods and is adaptable to high‐throughput imaging experiments. VoltageFluors, a class of voltage‐sensitive dyes, have recently been used to optically study the spontaneous activity of many neurons simultaneously in dissociated culture. VoltageFluors are particularly useful for experiments investigating differences in excitability and connectivity between neurons at different stages of development and in different disease models. The protocols in this article describe general procedures for preparing dissociated cultures, imaging spontaneous activity in dissociated cultures with VoltageFluors, and analyzing optical spontaneous activity data. © 2021 Wiley Periodicals LLC. This article was corrected on 20 July 2022. See the end of the full text for details. Basic Protocol 1: Preparation of dissociated rat hippocampal or cortical cultures Alternate Protocol: Preparation of microisland dissociated cultures Basic Protocol 2: Imaging of spontaneous activity in dissociated cultures using voltage‐sensitive dyes Basic Protocol 3: Analysis of spontaneous activity imaging datamore » « less
-
ABSTRACT The enteric nervous system is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and these are pertinent subjects for study due to their relevance to the human congenital disease Hirschsprung disease (HSCR). For the first time, we performed in toto gut imaging and single-cell generation tracing of ENCC migration in wild type and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs localized along the gut, and these ENCCs failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation-dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature in retwmr1/+ ENCCs. These results suggest that Ret signaling may regulate maintenance of a stem state in ENCCs.more » « less
-
Fluorescence microscopy and genetically encoded calcium indicators help understand brain function by recording large-scalein vivovideos in assorted animal models. Extracting the fluorescent transients that represent active periods of individual neurons is a key step when analyzing imaging videos. Non-specific calcium sources and background adjacent to segmented neurons contaminate the neurons’ temporal traces with false transients. We developed and characterized a novel method, temporal unmixing of calcium traces (TUnCaT), to quickly and accurately unmix the calcium signals of neighboring neurons and background. Our algorithm used background subtraction to remove the false transients caused by background fluctuations, and then applied targeted non-negative matrix factorization to remove the false transients caused by neighboring calcium sources. TUnCaT was more accurate than existing algorithms when processing multiple experimental and simulated datasets. TUnCaT’s speed was faster than or comparable to existing algorithms.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/science.aav6416
