scholarly journals Deformable mirror-based two-photon microscopy for axial mammalian brain imaging

2019 ◽  
Author(s):  
Alba Peinado ◽  
Eduardo Bendek ◽  
Sae Yokoyama ◽  
Kira E. Poskanzer
Keyword(s):  
Brain Imaging ◽  
High Speed ◽  
Brain Slices ◽  
Axial Position ◽  
Mammalian Brain ◽  
Deformable Mirror ◽  
Radius Of Curvature ◽  
Step Size ◽  
Two Photon

AbstractThis work presents the design and implementation of an enhanced version of a traditional two-photon (2P) microscope with the addition of high-speed axial scanning for live mammalian brain imaging. Our implementation utilizes a deformable mirror (DM) that can rapidly apply different defocus shapes to manipulate the laser beam divergence and consequently control the axial position of the beam focus in the sample. We provide a mathematical model describing the DM curvature, then experimentally characterize the radius of curvature as well as the Zernike terms of the DM surface for a given set of defocuses. A description of the optical setup of the 2P microscope is detailed. We conduct a thorough calibration of the system, determining the point spread function, the total scanning range, the axial step size, and the intensity curvature as a function of depth. Finally, the instrument is used for imaging different neurobiological samples, including fixed brain slices and in vivo mouse cerebral cortex.

scholarly journals Deformable mirror-based axial scanning for two-photon mammalian brain imaging

2021 ◽  
Vol 8 (01) ◽  
Author(s):  
Alba Peinado ◽  
Eduardo Bendek ◽  
Sae Yokoyama ◽  
Kira E. Poskanzer
Keyword(s):  
Brain Imaging ◽  
Mammalian Brain ◽  
Deformable Mirror ◽  
Two Photon ◽  
Axial Scanning

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 Identification of cellular-activity dynamics across large tissue volumes in the mammalian brain

2017 ◽  
Author(s):  
Logan Grosenick ◽  
Michael Broxton ◽  
Christina K. Kim ◽  
Conor Liston ◽  
Ben Poole ◽  
...  
Keyword(s):  
High Speed ◽  
Dorsal Hippocampus ◽  
Computational Imaging ◽  
Three Dimensions ◽  
Large Field ◽  
Mammalian Brain ◽  
Subcortical Structures ◽  
Intact Tissue ◽  
Context Learning

Tracking the coordinated activity of cellular events through volumes of intact tissue is a major challenge in biology that has inspired significant technological innovation. Yet scanless measurement of the high-speed activity of individual neurons across three dimensions in scattering mammalian tissue remains an open problem. Here we develop and validate a computational imaging approach (SWIFT) that integrates high-dimensional, structured statistics with light field microscopy to allow the synchronous acquisition of single-neuron resolution activity throughout intact tissue volumes as fast as a camera can capture images (currently up to 100 Hz at full camera resolution), attaining rates needed to keep pace with emerging fast calcium and voltage sensors. We demonstrate that this large field-of-view, single-snapshot volume acquisition method—which requires only a simple and inexpensive modification to a standard fluorescence microscope—enables scanless capture of coordinated activity patterns throughout mammalian neural volumes. Further, the volumetric nature of SWIFT also allows fast in vivo imaging, motion correction, and cell identification throughout curved subcortical structures like the dorsal hippocampus, where cellular-resolution dynamics spanning hippocampal subfields can be simultaneously observed during a virtual context learning task in a behaving animal. SWIFT’s ability to rapidly and easily record from volumes of many cells across layers opens the door to widespread identification of dynamical motifs and timing dependencies among coordinated cell assemblies during adaptive, modulated, or maladaptive physiological processes in neural systems.

scholarly journals Imaging Subthreshold Voltage Oscillation With Cellular Resolution in the Inferior Olive in vitro

2020 ◽  
Vol 14 ◽  
Author(s):  
Kevin Dorgans ◽  
Bernd Kuhn ◽  
Marylka Yoe Uusisaari
Keyword(s):  
Inferior Olive ◽  
Brain Slices ◽  
Brain Regions ◽  
Mammalian Brain ◽  
Wide Field ◽  
Voltage Imaging ◽  
Subthreshold Oscillations ◽  
Cellular Resolution

Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.

Ultrafast two-photon microscopy for high-speed brain imaging in awake mice (Conference Presentation)

2018 ◽  
Author(s):  
Radoslaw Chrapkiewicz ◽  
Tong Zhang ◽  
Oscar Hernandez ◽  
Adam S. Shai ◽  
Mark J. Wagner ◽  
...  
Keyword(s):  
Brain Imaging ◽  
High Speed ◽  
Two Photon Microscopy ◽  
Two Photon

NIR‐II Excitable Conjugated Polymer Dots with Bright NIR‐I Emission for Deep In Vivo Two‐Photon Brain Imaging Through Intact Skull

2019 ◽  
Vol 29 (15) ◽  
pp. 1808365 ◽  
Author(s):  
Shaowei Wang ◽  
Jie Liu ◽  
Guangxue Feng ◽  
Lai Guan Ng ◽  
Bin Liu
Keyword(s):  
Brain Imaging ◽  
Conjugated Polymer ◽  
Two Photon ◽  
Polymer Dots

scholarly journals A platform for brain-wide imaging and reconstruction of individual neurons

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Michael N Economo ◽  
Nathan G Clack ◽  
Luke D Lavis ◽  
Charles R Gerfen ◽  
Karel Svoboda ◽  
...  
Keyword(s):  
Long Range ◽  
High Speed ◽  
Large Scale ◽  
Three Dimensional ◽  
Image Data ◽  
Mammalian Brain ◽  
Projection Neurons ◽  
Computational Tools ◽  
Two Photon ◽  
Axonal Arbors

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.

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 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.

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