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

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.

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Dual-color volumetric imaging of neural activity of cortical columns

10.1101/504233 ◽

2018 ◽

Cited By ~ 1

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.

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

Biomedical Optics Express ◽

10.1364/boe.405738 ◽

2020 ◽

Vol 12 (1) ◽

pp. 162

Author(s):

Yu-Feng Chien ◽

Jyun-Yi Lin ◽

Po-Ting Yeh ◽

Kuo-Jen Hsu ◽

Yu-Hsuan Tsai ◽

...

Keyword(s):

Brain Imaging ◽

High Speed ◽

Two Photon ◽

Grin Lens ◽

Deep Brain

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High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

Nature Communications ◽

10.1038/s41467-020-19851-1 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 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.

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The Power of Single and Multibeam Two-Photon Microscopy for High-Resolution and High-Speed Deep Tissue and Intravital Imaging

Biophysical Journal ◽

10.1529/biophysj.106.102459 ◽

2007 ◽

Vol 93 (7) ◽

pp. 2519-2529 ◽

Cited By ~ 66

Author(s):

Raluca Niesner ◽

Volker Andresen ◽

Jens Neumann ◽

Heinrich Spiecker ◽

Matthias Gunzer

Keyword(s):

High Resolution ◽

High Speed ◽

Intravital Imaging ◽

Deep Tissue ◽

Two Photon Microscopy ◽

Two Photon

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

10.7554/elife.40805.019 ◽

2018 ◽

Keyword(s):

High Throughput ◽

Volumetric Imaging ◽

Two Photon ◽

Two Photon Fluorescence ◽

Photon Fluorescence ◽

Deep Brain

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Adaptive optics two-photon endomicroscopy enables deep-brain imaging at synaptic resolution over large volumes

Science Advances ◽

10.1126/sciadv.abc6521 ◽

2020 ◽

Vol 6 (40) ◽

pp. eabc6521 ◽

Cited By ~ 1

Author(s):

Zhongya Qin ◽

Congping Chen ◽

Sicong He ◽

Ye Wang ◽

Kam Fai Tam ◽

...

Keyword(s):

Brain Imaging ◽

Adaptive Optics ◽

Critical Role ◽

Random Access ◽

Brain Structures ◽

Invasive Approach ◽

Two Photon ◽

Grin Lens ◽

Deep Brain

Optical deep-brain imaging in vivo at high resolution has remained a great challenge over the decades. Two-photon endomicroscopy provides a minimally invasive approach to image buried brain structures, once it is integrated with a gradient refractive index (GRIN) lens embedded in the brain. However, its imaging resolution and field of view are compromised by the intrinsic aberrations of the GRIN lens. Here, we develop a two-photon endomicroscopy by adding adaptive optics based on direct wavefront sensing, which enables recovery of diffraction-limited resolution in deep-brain imaging. A new precompensation strategy plays a critical role to correct aberrations over large volumes and achieve rapid random-access multiplane imaging. We investigate the neuronal plasticity in the hippocampus, a critical deep brain structure, and reveal the relationship between the somatic and dendritic activity of pyramidal neurons.

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Deep tissue scattering compensation with three-photon F-SHARP

10.1101/2021.08.18.456049 ◽

2021 ◽

Author(s):

Caroline Berlage ◽

Malinda L. S. Tantirigama ◽

Mathias Babot ◽

Diego Di Battista ◽

Clarissa Whitmire ◽

...

Keyword(s):

Penetration Depth ◽

Dendritic Spines ◽

Imaging Techniques ◽

Biological Research ◽

Biological Applications ◽

Deep Tissue ◽

Two Photon ◽

Tissue Scattering ◽

Compensation Technique ◽

Wavefront Shaping

Optical imaging techniques are widely used in biological research, but their penetration depth is limited by tissue scattering. Wavefront shaping techniques are able to overcome this problem in principle, but are often slow and their performance depends on the sample. This greatly reduces their practicability for biological applications. Here we present a scattering compensation technique based on three-photon (3P) excitation, which converges faster than comparable two-photon (2P) techniques and works reliably even on densely labeled samples, where 2P approaches fail. To demonstrate its usability and advantages for biomedical imaging we apply it to the imaging of dendritic spines on GFP-labeled layer 5 neurons in an anesthetized mouse.

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Optical clearing of living brains with MAGICAL to extend in vivo imaging

10.1101/507426 ◽

2019 ◽

Cited By ~ 2

Author(s):

Kouichirou Iijima ◽

Takuto Oshima ◽

Ryosuke Kawakami ◽

Tomomi Nemoto

Keyword(s):

Fluorescence Emission ◽

Cortical Layer ◽

Fluorescence Emission Spectrum ◽

Spatiotemporal Resolution ◽

Two Photon Microscopy ◽

Two Photon ◽

Brain Functions ◽

Hippocampal Ca1 ◽

Layer V

AbstractTo understand brain functions, it is important to observe directly how multiple neural circuits are performing in living brains. However, due to tissue opaqueness, observable depth and spatiotemporal resolution are severely degraded in vivo. Here, we propose an optical brain clearing method for in vivo fluorescence microscopy, termed MAGICAL (Magical Additive Glycerol Improves Clear Alive Luminance). MAGICAL enabled two-photon microscopy to capture vivid images with fast speed, at cortical layer V and hippocampal CA1 in vivo. Moreover, MAGICAL promoted conventional confocal microscopy to visualize finer neuronal structures including synaptic boutons and spines in unprecedented deep regions, without intensive illumination leading to phototoxic effects. Fluorescence Emission Spectrum Transmissive Analysis (FESTA) showed that MAGICAL improved in vivo transmittance of shorter wavelength light, which is vulnerable to optical scattering thus unsuited for in vivo microscopy. These results suggest that MAGICAL would transparentize living brains via scattering reduction.

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Fast two-photon volumetric imaging of an improved voltage indicator reveals electrical activity in deeply located neurons in the awake brain

10.1101/445064 ◽

2018 ◽

Cited By ~ 14

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.

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