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

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.

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

10.1101/2021.10.15.464407 ◽

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.

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Three-Dimensional Structural Characterization of Nonwoven Fabrics

Microscopy and Microanalysis ◽

10.1017/s143192761201375x ◽

2012 ◽

Vol 18 (6) ◽

pp. 1368-1379 ◽

Cited By ~ 10

Author(s):

Lalith B. Suragani Venu ◽

Eunkyoung Shim ◽

Nagendra Anantharamaiah ◽

Behnam Pourdeyhimi

Keyword(s):

High Speed ◽

Imaging Technique ◽

Low Cost ◽

Three Dimensional ◽

Complex Nature ◽

Volumetric Imaging ◽

Analysis Techniques ◽

Nonwoven Fabrics ◽

Nonwoven Materials

AbstractNonwoven materials are found in a gamut of critical applications. This is partly due to the fact that these structures can be produced at high speed and engineered to deliver unique functionality at low cost. The behavior of these materials is highly dependent on alignment of fibers within the structure. The ability to characterize and also to control the structure is important, but very challenging due to the complex nature of the structures. Thus, to date, focus has been placed mainly on two-dimensional analysis techniques for describing the behavior of nonwovens. This article demonstrates the utility of three-dimensional (3D) digital volumetric imaging technique for visualizing and characterizing a complex 3D class of nonwoven structures produced by hydroentanglement.

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

Membranes ◽

10.3390/membranes11080634 ◽

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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Light-Sheet Microscopy in Neuroscience

Annual Review of Neuroscience ◽

10.1146/annurev-neuro-070918-050357 ◽

2019 ◽

Vol 42 (1) ◽

pp. 295-313 ◽

Cited By ~ 32

Author(s):

Elizabeth M.C. Hillman ◽

Venkatakaushik Voleti ◽

Wenze Li ◽

Hang Yu

Keyword(s):

High Speed ◽

Light Sheet ◽

Mammalian Brain ◽

Diverse Range ◽

Volumetric Imaging ◽

Two Photon Microscopy ◽

Light Sheet Microscopy ◽

Noise Benefits ◽

Longitudinal Imaging

Light-sheet microscopy is an imaging approach that offers unique advantages for a diverse range of neuroscience applications. Unlike point-scanning techniques such as confocal and two-photon microscopy, light-sheet microscopes illuminate an entire plane of tissue, while imaging this plane onto a camera. Although early implementations of light sheet were optimized for longitudinal imaging of embryonic development in small specimens, emerging implementations are capable of capturing light-sheet images in freely moving, unconstrained specimens and even the intact in vivo mammalian brain. Meanwhile, the unique photobleaching and signal-to-noise benefits afforded by light-sheet microscopy's parallelized detection deliver the ability to perform volumetric imaging at much higher speeds than can be achieved using point scanning. This review describes the basic principles and evolution of light-sheet microscopy, followed by perspectives on emerging applications and opportunities for both imaging large, cleared, and expanded neural tissues and high-speed, functional imaging in vivo.

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