scholarly journals Fast two-photon volumetric imaging of an improved voltage indicator reveals electrical activity in deeply located neurons in the awake brain

2018 ◽  
Author(s):  
Mariya Chavarha ◽  
Vincent Villette ◽  
Ivan K. Dimov ◽  
Lagnajeet Pradhan ◽  
Stephen W. Evans ◽  
...  
Keyword(s):  
Electrical Activity ◽  
High Speed ◽  
Scanning Method ◽  
Volumetric Imaging ◽  
Voltage Range ◽  
Two Photon ◽  
Repeated Sampling ◽  
Visual Cortex Neurons ◽  
Voltage Indicator

ABSTRACTImaging of transmembrane voltage deep in brain tissue with cellular resolution has the potential to reveal information processing by neuronal circuits in living animals with minimal perturbation. Multi-photon voltage imaging in vivo, however, is currently limited by speed and sensitivity of both indicators and imaging methods. Here, we report the engineering of an improved genetically encoded voltage indicator, ASAP3, which exhibits up to 51% fluorescence responses in the physiological voltage range, sub-millisecond activation kinetics, and full responsivity under two-photon illumination. We also introduce an ultrafast local volume excitation (ULOVE) two-photon scanning method to sample ASAP3 signals in awake mice at kilohertz rates with increased stability and sensitivity. ASAP3 and ULOVE allowed continuous single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution, and with repeated sampling over multiple days. By imaging voltage in visual cortex neurons, we found evidence for cell type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULOVE enable continuous high-speed high-resolution imaging of electrical activity in deeply located genetically defined neurons during awake behavior.

scholarly journals Dual-color volumetric imaging of neural activity of cortical columns

2018 ◽  
Author(s):  
Shuting Han ◽  
Weijian Yang ◽  
Rafael Yuste
Keyword(s):  
Neural Activity ◽  
High Speed ◽  
Neural Circuits ◽  
Emergent Properties ◽  
Spatiotemporal Resolution ◽  
Population Activity ◽  
Volumetric Imaging ◽  
Two Photon ◽  
Dual Color

To capture the emergent properties of neural circuits, high-speed volumetric imaging of neural activity at cellular resolution is desirable. But while conventional two-photon calcium imaging is a powerful tool to study population activity in vivo, it is restrained to two-dimensional planes. Expanding it to 3D while maintaining high spatiotemporal resolution appears necessary. Here, we developed a two-photon microscope with dual-color laser excitation that can image neural activity in a 3D volume. We imaged the neuronal activity of primary visual cortex from awake mice, spanning from L2 to L5 with 10 planes, at a rate of 10 vol/sec, and demonstrated volumetric imaging of L1 long-range PFC projections and L2/3 somatas. Using this method, we map visually-evoked neuronal ensembles in 3D, finding a lack of columnar structure in orientation responses and revealing functional correlations between cortical layers which differ from trial to trial and are missed in sequential imaging. We also reveal functional interactions between presynaptic L1 axons and postsynaptic L2/3 neurons. Volumetric two-photon imaging appears an ideal method for functional connectomics of neural circuits.

scholarly journals High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiang Lan Fan ◽  
Jose A. Rivera ◽  
Wei Sun ◽  
John Peterson ◽  
Henry Haeberle ◽  
...  
Keyword(s):  
Blood Flow ◽  
High Speed ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Fluorescence Signal ◽  
Imaging Method ◽  
Spatiotemporal Resolution ◽  
Two Photon

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.

High speed UHR-OCT for in-vivo volumetric imaging of the palisades of Vogt and the cellular structure of the limbal crypts in the healthy and pathological human corneo-scleral limbus (Conference Presentation)

2018 ◽  
Author(s):  
Kostadinka Bizheva ◽  
Bingyao Tan ◽  
Zohreh Hosseinaee ◽  
Kirsten Carter ◽  
Denise Hileeto ◽  
...  
Keyword(s):  
Cellular Structure ◽  
High Speed ◽  
Volumetric Imaging

In-vivo volumetric imaging of the cellular structure of healthy and pathological human cornea with high-speed UHR-OCT (Conference Presentation)

2018 ◽  
Author(s):  
Zohreh Hosseinaee ◽  
Bingyao Tan ◽  
Kirsten Carter ◽  
Denise Hileeto ◽  
Luigina Sorbara ◽  
...  
Keyword(s):  
Cellular Structure ◽  
High Speed ◽  
Human Cornea ◽  
Volumetric Imaging

scholarly journals A Multifocal Multiphoton Volumetric Imaging Technique for High Speed Time-Resolved FRET Imaging in vivo

2016 ◽  
Vol 110 (3) ◽  
pp. 165a ◽  
Author(s):  
Simon P. Poland ◽  
James A. Levitt ◽  
Nikola Krstajić ◽  
Ahmet Erdogen ◽  
Richard J. Walker ◽  
...  
Keyword(s):  
High Speed ◽  
Imaging Technique ◽  
Time Resolved ◽  
Volumetric Imaging

scholarly journals Single-Shot Optical Projection tomography for high-speed volumetric imaging

2021 ◽  
Author(s):  
Connor James Darling ◽  
Samuel P.X. Davis ◽  
Sunil Kumar ◽  
Paul M.W. French ◽  
James A McGinty
Keyword(s):  
High Speed ◽  
Low Cost ◽  
Single Shot ◽  
Optical Projection Tomography ◽  
Data Set ◽  
Volumetric Imaging ◽  
Optical Projection ◽  
Volumetric Reconstruction ◽  
Projection Images

We present a single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic samples. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio but the sequential acquisition of projection images required for volumetric reconstruction typically requires samples to be immobilised during the acquisition of an OPT data set. We present a proof-of-principle system capable of single-shot imaging of a 1 mm diameter volume, demonstrating camera-limited rates of up to 62.5 volumes/second, which we have applied to 3D imaging of a freely-swimming zebrafish embryo. This is achieved by recording 8 projection views simultaneously on 4 low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under 5,000GBP. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including fluid dynamics.

scholarly journals Two-Photon Imaging of Electrical Activity in Mouse Cortex using a Genetically-Encoded Voltage Indicator

2013 ◽  
Vol 104 (2) ◽  
pp. 336a
Author(s):  
Walther Akemann ◽  
Hiroki Mutoh ◽  
Thomas Knöpfel
Keyword(s):  
Electrical Activity ◽  
Two Photon ◽  
Mouse Cortex ◽  
Photon Imaging ◽  
Two Photon Imaging ◽  
Voltage Indicator

scholarly journals Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy

2011 ◽  
Vol 105 (6) ◽  
pp. 3106-3113 ◽  
Author(s):  
Jonathan D. Driscoll ◽  
Andy Y. Shih ◽  
Satish Iyengar ◽  
Jeffrey J. Field ◽  
G. Allen White ◽  
...  
Keyword(s):  
High Speed ◽  
Signal To Noise Ratio ◽  
Photon Counting ◽  
In Vivo Model ◽  
Fluorescence Lifetime Imaging ◽  
Photon Counter ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Lifetime Imaging

We present a high-speed photon counter for use with two-photon microscopy. Counting pulses of photocurrent, as opposed to analog integration, maximizes the signal-to-noise ratio so long as the uncertainty in the count does not exceed the gain-noise of the photodetector. Our system extends this improvement through an estimate of the count that corrects for the censored period after detection of an emission event. The same system can be rapidly reconfigured in software for fluorescence lifetime imaging, which we illustrate by distinguishing between two spectrally similar fluorophores in an in vivo model of microstroke.

scholarly journals HiLo Based Line Scanning Temporal Focusing Microscopy for High-Speed, Deep Tissue Imaging

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 634
Author(s):  
Ruheng Shi ◽  
Yuanlong Zhang ◽  
Tiankuang Zhou ◽  
Lingjie Kong
Keyword(s):  
High Speed ◽  
Three Dimensions ◽  
Tissue Imaging ◽  
Deep Penetration ◽  
High Temporal Resolution ◽  
Structured Illumination ◽  
Volumetric Imaging ◽  
Temporal Focusing ◽  
Line Scanning

High-speed, optical-sectioning imaging is highly desired in biomedical studies, as most bio-structures and bio-dynamics are in three-dimensions. Compared to point-scanning techniques, line scanning temporal focusing microscopy (LSTFM) is a promising method that can achieve high temporal resolution while maintaining a deep penetration depth. However, the contrast and axial confinement would still be deteriorated in scattering tissue imaging. Here, we propose a HiLo-based LSTFM, utilizing structured illumination to inhibit the fluorescence background and, thus, enhance the image contrast and axial confinement in deep imaging. We demonstrate the superiority of our method by performing volumetric imaging of neurons and dynamical imaging of microglia in mouse brains in vivo.

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