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.

scholarly journals Precise, 3-D optogenetic control of the diameter of single arterioles

2021 ◽  
Author(s):  
Philip J. O’Herron ◽  
David A. Hartmann ◽  
Kun Xie ◽  
Prakash Kara ◽  
Andy Y. Shih
Keyword(s):  
Blood Flow ◽  
Neural Activity ◽  
Light Modulator ◽  
Spatiotemporal Resolution ◽  
Two Photon ◽  
Light Pulses ◽  
All Optical ◽  
Health And Disease ◽  
And Control

AbstractModulation of brain arteriole diameter is critical for maintenance of cerebral blood pressure and control of hyperemia during regional neural activity. However, studies of hemodynamic function in health and disease have lacked a method to control and monitor blood flow with high spatiotemporal resolution. Here, we describe a new all-optical approach to precisely control and monitor arteriolar contractility in vivo using combined two-photon optogenetics and imaging. The expression of the excitatory opsin, ReaChR, in vascular smooth muscle cells enabled rapid and repeated vasoconstriction following brief light pulses. Targeted two-photon activation of ReaCHR using a spatial light modulator (SLM) produced highly localized constrictions when targeted to individual arteries within the neocortex. We demonstrate the utility of this method for examining arteriole contractile dynamics and creating transient blood flow reductions. Additionally, we show that optogenetic constriction can offset or completely block sensory stimulus evoked vasodilation, providing a valuable tool to dissociate blood flow changes from neural activity.

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 Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo

2019 ◽  
Author(s):  
Jianglai Wu ◽  
Yajie Liang ◽  
Shuo Chen ◽  
Ching-Lung Hsu ◽  
Mariya Chavarha ◽  
...  
Keyword(s):  
Neural Activity ◽  
Laser Scanning ◽  
Imaging Method ◽  
Spatiotemporal Resolution ◽  
Microscopy Imaging ◽  
Two Photon ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence ◽  
The Brain

Understanding information processing in the brain requires us to monitor neural activity in vivo at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope (2PFM) empowered by all-optical laser scanning, we imaged neural activity in vivo at up to 3,000 frames per second and submicron spatial resolution. This ultrafast imaging method enabled monitoring of both supra- and sub-threshold electrical activity down to 345 μm below the brain surface in head fixed awake mice.

scholarly journals Three-dimensional Two-Photon Optogenetics and Imaging of Neural Circuits in vivo

2017 ◽  
Author(s):  
Weijian Yang ◽  
Luis Carrillo-Reid ◽  
Yuki Bando ◽  
Darcy S. Peterka ◽  
Rafael Yuste
Keyword(s):  
Visual Cortex ◽  
Neural Activity ◽  
Calcium Imaging ◽  
Pyramidal Neurons ◽  
Neural Circuits ◽  
Three Dimensional ◽  
Two Photon ◽  
Cellular Resolution ◽  
Photon Excitation

We demonstrate a holographic system for simultaneous three-dimensional (3D) two-photon stimulation and imaging of neural activity in the mouse neocortex in vivo with cellular resolution. Dual two-photon excitation paths are implemented with independent 3D targeting for calcium imaging and precision optogenetics. We validate the usefulness of the microscope by photoactivating local pools of interneurons in awake mice visual cortex in 3D, which suppress the nearby pyramidal neurons’ response to visual stimuli.

Dual-color volumetric imaging of neural activity of cortical columns in vivo (Conference Presentation)

2019 ◽  
Author(s):  
Shuting Han ◽  
Weijian Yang ◽  
Rafael Yuste
Keyword(s):  
Neural Activity ◽  
Volumetric Imaging ◽  
Cortical Columns ◽  
Dual Color

scholarly journals Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging

2020 ◽  
Author(s):  
Yu-Feng Chien ◽  
Jyun-Yi Lin ◽  
Po-Ting Yeh ◽  
Kuo-Jen Hsu ◽  
Yu-Hsuan Tsai ◽  
...  
Keyword(s):  
Penetration Depth ◽  
High Speed ◽  
Gradient Index ◽  
Spatiotemporal Resolution ◽  
Deep Tissue ◽  
Volumetric Imaging ◽  
Two Photon ◽  
Brain Functions ◽  
Grin Lens ◽  
Deep Brain

AbstractStudying neural connections and activities in vivo is fundamental to understanding brain functions. Given the cm-size brain and three-dimensional neural circuit dynamics, deep-tissue, high-speed volumetric imaging is highly desirable for brain study. With sub-micrometer spatial resolution, intrinsic optical sectioning, and deep-tissue penetration capability, two-photon microscopy (2PM) has found a niche in neuroscience. However, current 2PM typically relies on slow axial scan for volumetric imaging, and the maximal penetration depth is only about 1 mm. Here, we demonstrate that by integrating two gradient-index (GRIN) lenses into 2PM, both penetration depth and volume-imaging rate can be significantly improved. Specifically, an 8-mm long GRIN lens allows imaging relay through a whole mouse brain, while a tunable acoustic gradient-index (TAG) lens provides sub-second volume rate via 100 kHz ∼ 1 MHz axial scan. This technique enables the study of calcium dynamics in cm-deep brain regions with sub-cellular and sub-second spatiotemporal resolution, paving the way for interrogating deep-brain functional connectome.

scholarly journals A high-speed, bright, red fluorescent voltage sensor to detect neural activity

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Connor Beck ◽  
Yiyang Gong
Keyword(s):  
Blue Light ◽  
Neural Activity ◽  
High Speed ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Fluorescent Sensors ◽  
Fast Kinetics ◽  
Spectral Separation ◽  
Green Fluorescent

Abstract Genetically encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy. GEVIs have demonstrated ultra-fast kinetics and high spike detection fidelity in vivo, but existing red-fluorescent voltage indicators fall short of the response and brightness achieved by green fluorescent protein-based sensors. Furthermore, red-fluorescent GEVIs suffer from incomplete spectral separation from green sensors and blue-light-activated optogenetic actuators. We have developed Ace-mScarlet, a red fluorescent GEVI that fuses Ace2N, a voltage-sensitive inhibitory rhodopsin, with mScarlet, a bright red fluorescent protein (FP). Through fluorescence resonance energy transfer (FRET), our sensor detects changes in membrane voltage with high sensitivity and brightness and has kinetics comparable to the fastest green fluorescent sensors. Ace-mScarlet’s red-shifted absorption and emission spectra facilitate virtually complete spectral separation when used in combination with green-fluorescent sensors or with blue-light-sensitive sensors and rhodopsins. This spectral separation enables both simultaneous imaging in two separate wavelength channels and high-fidelity voltage recordings during simultaneous optogenetic perturbation.

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
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