Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

High-throughput deep tissue two-photon microscopy at kilohertz frame rates.

ABSTRACT: High-speed laser scanning microscopes are essential for monitoring fast biological phenomena. However, existing strategies that achieve millisecond time resolution with two-photon microscopes (2PMs) are generally technically challenging and suffer from compromises among imaging field of view, excitation efficiency, and depth penetration in thick tissue. Here, we present a versatile solution that enables a conventional video-rate 2PM to perform 2D scanning at kilohertz frame rates over large fields of view. Our system is based on implementation of a scan multiplier unit that provides inertia-free multiplication of the scanning speed while preserving all the benefits of standard 2PM. We demonstrate kilohertz subcellular-resolution 2PM imaging with an order of magnitude higher imaging throughput than previously achievable and penetration depths exceeding 500 μm, which we apply to the study of neurovascular coupling dynamics in the mouse brain.

SUBMITTER: Xiao S

PROVIDER: S-EPMC11178336 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

High-throughput deep tissue two-photon microscopy at kilohertz frame rates.

Xiao Sheng S Xiao Sheng S Giblin John T JT Boas David A DA Mertz Jerome J

Optica 20230615 6

High-speed laser scanning microscopes are essential for monitoring fast biological phenomena. However, existing strategies that achieve millisecond time resolution with two-photon microscopes (2PMs) are generally technically challenging and suffer from compromises among imaging field of view, excitation efficiency, and depth penetration in thick tissue. Here, we present a versatile solution that enables a conventional video-rate 2PM to perform 2D scanning at kilohertz frame rates over large fiel ...[more]

PMID: 38882052

Similar Datasets

Kilohertz frame-rate two-photon tomography.

Project description:Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static 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 anesthetized mice are synchronized within spatially contiguous domains spanning tens of micrometers at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, three-dimensional single-particle tracking and imaging in densely labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

| S-EPMC6754705 | biostudies-literature

Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates.

Project description:Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) - microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.

| S-EPMC7188897 | biostudies-literature

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

Project description:Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.

| S-EPMC7199528 | biostudies-literature

Single Photon Kilohertz Frame Rate Imaging of Neural Activity.

Project description:Establishing the biological basis of cognition and its disorders will require high precision spatiotemporal measurements of neural activity. Recently developed genetically encoded voltage indicators (GEVIs) report both spiking and subthreshold activity of identified neurons. However, maximally capitalizing on the potential of GEVIs will require imaging at millisecond time scales, which remains challenging with standard camera systems. Here, application of single photon avalanche diode (SPAD) sensors is reported to image neural activity at kilohertz frame rates. SPADs are electronic devices that when activated by a single photon cause an avalanche of electrons and a large electric current. An array of SPAD sensors is used to image individual neurons expressing the GEVI Voltron-JF525-HTL. It is shown that subthreshold and spiking activity can be resolved with shot noise limited signals at frame rates of up to 10 kHz. SPAD imaging is able to reveal millisecond scale synchronization of neural activity in an ex vivo seizure model. SPAD sensors may have widespread applications for investigation of millisecond timescale neural dynamics.

| S-EPMC9631062 | biostudies-literature

The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.

Project description:Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.

| S-EPMC1965440 | biostudies-literature

High throughput detection of capillary stalling events with Bessel beam two-photon microscopy.

Project description:SignificanceBrief disruptions in capillary flow, commonly referred to as capillary "stalling," have gained interest recently for their potential role in disrupting cerebral blood flow and oxygen delivery. Approaches to studying this phenomenon have been hindered by limited volumetric imaging rates and cumbersome manual analysis. The ability to precisely and efficiently quantify the dynamics of these events will be key in understanding their potential role in stroke and neurodegenerative diseases, such as Alzheimer's disease.AimOur study aimed to demonstrate that the fast volumetric imaging rates offered by Bessel beam two-photon microscopy combined with improved data analysis throughput allows for faster and more precise measurement of capillary stall dynamics.ResultsWe found that while our analysis approach was unable to achieve full automation, we were able to cut analysis time in half while also finding stalling events that were missed in traditional blind manual analysis. The resulting data showed that our Bessel beam system was captured more stalling events compared to optical coherence tomography, particularly shorter stalling events. We then compare differences in stall dynamics between a young and old group of mice as well as a demonstrate changes in stalling before and after photothrombotic model of stroke. Finally, we also demonstrate the ability to monitor arteriole dynamics alongside stall dynamics.ConclusionsBessel beam two-photon microscopy combined with high throughput analysis is a powerful tool for studying capillary stalling due to its ability to monitor hundreds of capillaries simultaneously at high frame rates.

| S-EPMC10495839 | biostudies-literature

Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy.

Project description:Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 μm and report voltage correlations in pairs of neurons.

| S-EPMC9563101 | biostudies-literature

Kilohertz two-photon brain imaging in awake mice.

Project description:Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10-30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000-211,000 µm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.

| S-EPMC9438750 | biostudies-literature

Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics <i>in vivo</i>.

Project description:SignificanceThe studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device.AimWe introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400 kHz and a maximum frame rate of 10,000 frames per second (fps) at 250×40 pixels . For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning.ApproachWith this combinatorial device that supports both large-view navigation and small-view high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs).ResultsWe measured dendritic calcium propagation velocity ( 80/62.5-116.7 μm/ms ) in OGB-1-labeled single cortical neurons in mice in vivo. To benchmark the spatial precision and detection sensitivity of measurement in vivo, we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille's law of laminar flow.ConclusionsThis proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo.

| S-EPMC10156610 | biostudies-literature

High-throughput synapse-resolving two-photon fluorescence microendoscopy for deep-brain volumetric imaging in vivo.

Project description:Optical imaging has become a powerful tool for studying brains in vivo. The opacity of adult brains makes microendoscopy, with an optical probe such as a gradient index (GRIN) lens embedded into brain tissue to provide optical relay, the method of choice for imaging neurons and neural activity in deeply buried brain structures. Incorporating a Bessel focus scanning module into two-photon fluorescence microendoscopy, we extended the excitation focus axially and improved its lateral resolution. Scanning the Bessel focus in 2D, we imaged volumes of neurons at high-throughput while resolving fine structures such as synaptic terminals. We applied this approach to the volumetric anatomical imaging of dendritic spines and axonal boutons in the mouse hippocampus, and functional imaging of GABAergic neurons in the mouse lateral hypothalamus in vivo.

| S-EPMC6338462 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data