scholarly journals High-Speed Low-Light In Vivo Two-Photon Voltage Imaging of Large Neuronal Populations

2021 ◽  
Author(s):  
Jelena Platisa ◽  
Xin Ye ◽  
Allison M Ahrens ◽  
Chang Liu ◽  
Ichun A Chen ◽  
...  
Keyword(s):  
Shot Noise ◽  
High Speed ◽  
Neural Circuit ◽  
Frame Rate ◽  
Tissue Imaging ◽  
Photon Flux ◽  
Voltage Imaging ◽  
Neuronal Populations ◽  
Two Photon

Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4x0.4mm2 field of view, and; a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise limited signals. Through these combined advances, we achieved simultaneous high-speed, deep-tissue imaging of more than one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.

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 Ultrabright red AIEgens for two-photon vascular imaging with high resolution and deep penetration

2018 ◽  
Vol 9 (10) ◽  
pp. 2705-2710 ◽  
Author(s):  
Wei Qin ◽  
Pengfei Zhang ◽  
Hui Li ◽  
Jacky W. Y. Lam ◽  
Yuanjing Cai ◽  
...  
Keyword(s):  
High Resolution ◽  
Vascular Imaging ◽  
Tissue Imaging ◽  
Deep Penetration ◽  
High Contrast ◽  
Deep Tissue ◽  
Two Photon ◽  
High Penetration ◽  
Deep Tissue Imaging

A successful strategy for the design of ultrabright red luminogens with aggregation-induced emission (AIE) features is reported. The AIE dots can be utilized as efficient fluorescent probes for in vivo deep-tissue imaging with high penetration depth and high contrast.

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 All-optical interrogation of neural circuits in behaving mice

2021 ◽  
Author(s):  
Lloyd E. Russell ◽  
Henry W.P. Dalgleish ◽  
Rebecca Nutbrown ◽  
Oliver Gauld ◽  
Dustin Herrmann ◽  
...  
Keyword(s):  
Neural Circuits ◽  
Neural Circuit ◽  
Barrel Cortex ◽  
Brain Area ◽  
Imaging Data ◽  
Temporal Precision ◽  
Two Photon ◽  
All Optical ◽  
The Impact

Recent advances combining two-photon calcium imaging and two-photon optogenetics with digital holography now allow us to read and write neural activity in vivo at cellular resolution with millisecond temporal precision. Such 'all-optical' techniques enable experimenters to probe the impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This protocol describes the experimental strategy and workflow for successful completion of typical all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system, the preparation of an indicator and opsin-expressing and task-performing animal, the characterization of functional and photostimulation responses and the design and implementation of an all-optical experiment. We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy using all-optical experiments in three different brain areas - barrel cortex, visual cortex and hippocampus - using different experimental setups. This approach can in principle be adapted to any brain area for all-optical interrogation experiments to probe functional connectivity in neural circuits and for investigating the relationship between neural circuit activity and behaviour.

scholarly journals Spatial integration during active tactile sensation drives elementary shape perception

2020 ◽  
Author(s):  
Jennifer Brown ◽  
Ian Antón Oldenburg ◽  
Gregory I. Telian ◽  
Sandon Griffin ◽  
Mieke Voges ◽  
...  
Keyword(s):  
High Speed ◽  
Neural Circuit ◽  
Barrel Cortex ◽  
Object Identification ◽  
Surface Orientation ◽  
Orientation Discrimination ◽  
Spatial Integration ◽  
Two Photon ◽  
Photon Imaging ◽  
Two Photon Imaging

SummaryActive haptic sensation is critical for object identification and manipulation, such as for tool use in humans, or prey capture in rodents. The neural circuit basis for recognizing objects through active touch alone is poorly understood. To address this gap, we combined optogenetics, two photon imaging, and high-speed behavioral tracking in mice solving a novel surface orientation discrimination task with their whiskers. We found that orientation discrimination required animals to summate input from multiple whiskers specifically along the whisker arc. Many animals discriminated the orientation of the stimulus per se, as their performance was invariant to the specific location of the presented stimulus. Two photon imaging showed that populations of neurons in the barrel cortex encoded each of the discriminated orientations, and this coding depended on integration over the whisker array. Finally, acute optogenetic inactivation of the barrel cortex strongly impaired surface orientation discrimination, and even cell-type specific optogenetic suppression of layer 4 excitatory neurons degraded performance, implying a role for superficial layers in this computation. These data suggest a model in which spatial summation over an active haptic array generates representations of an object’s surface orientations. These computations may facilitate the encoding of complex three-dimensional objects during active exploration.

scholarly journals Kilohertz frame-rate two-photon tomography

2018 ◽  
Author(s):  
Abbas Kazemipour ◽  
Ondrej Novak ◽  
Daniel Flickinger ◽  
Jonathan S. Marvin ◽  
Jonathan King ◽  
...  
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Glutamate Release ◽  
Single Particle Tracking ◽  
Frame Rate ◽  
Mammalian Brain ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Calcium Sensors ◽  
Structural Image

SummaryPoint-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

scholarly journals Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

2021 ◽  
Author(s):  
Ahmed S Abdelfattah ◽  
Jihong Zheng ◽  
Daniel Reep ◽  
Getahun Tsegaye ◽  
Arthur Tsang ◽  
...  
Keyword(s):  
Saturation Mutagenesis ◽  
Voltage Sensitivity ◽  
Voltage Imaging ◽  
Neuronal Populations ◽  
Lower Baseline ◽  
Transmembrane Voltage ◽  
Automated Patch Clamp ◽  
Subthreshold Voltage ◽  
Voltage Indicator

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased ΔF/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

In vivo mucosal tissue imaging using fiber-based two-photon approach

2007 ◽  
Author(s):  
Xudong Xiao ◽  
Shilagard Tuya ◽  
Gracie Vargas
Keyword(s):  
Tissue Imaging ◽  
Mucosal Tissue ◽  
Two Photon

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 In Vivo Mammalian Brain Imaging Using One- and Two-Photon Fluorescence Microendoscopy

2004 ◽  
Vol 92 (5) ◽  
pp. 3121-3133 ◽  
Author(s):  
Juergen C. Jung ◽  
Amit D. Mehta ◽  
Emre Aksay ◽  
Raymond Stepnoski ◽  
Mark J. Schnitzer
Keyword(s):  
Laser Scanning ◽  
Frame Rate ◽  
Mammalian Brain ◽  
Cell Dynamics ◽  
Ca1 Neurons ◽  
Two Photon ◽  
Two Photon Fluorescence ◽  
Rats And Mice ◽  
Photon Fluorescence

One of the major limitations in the current set of techniques available to neuroscientists is a dearth of methods for imaging individual cells deep within the brains of live animals. To overcome this limitation, we developed two forms of minimally invasive fluorescence microendoscopy and tested their abilities to image cells in vivo. Both one- and two-photon fluorescence microendoscopy are based on compound gradient refractive index (GRIN) lenses that are 350–1,000 μm in diameter and provide micron-scale resolution. One-photon microendoscopy allows full-frame images to be viewed by eye or with a camera, and is well suited to fast frame-rate imaging. Two-photon microendoscopy is a laser-scanning modality that provides optical sectioning deep within tissue. Using in vivo microendoscopy we acquired video-rate movies of thalamic and CA1 hippocampal red blood cell dynamics and still-frame images of CA1 neurons and dendrites in anesthetized rats and mice. Microendoscopy will help meet the growing demand for in vivo cellular imaging created by the rapid emergence of new synthetic and genetically encoded fluorophores that can be used to label specific brain areas or cell classes.

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