Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain

Wang, Tianyu; Wu, Chunyan; Ouzounov, Dimitre G.; Gu, Wenchao; Xia, Fei; Kim, Minsu; Yang, Xusan; Warden, Melissa R.; Xu, Chris

doi:10.7554/elife.53205

Cited by 91 publications

(100 citation statements)

References 51 publications

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“…Photon trajectories, light intensities and tissue heating were calculated using a Monte Carlo random-walk model implemented in Python. The model was almost identical to that in several previous studies ( Wang et al, 1995 ; Stujenske et al, 2015 ; Podgorski and Ranganathan, 2016 ; Wang et al, 2020 ). Individual photons or packets of photons moved stochastically through the 3-dimensional volume, in which they were subjected to absorption and scattering by the tissue.…”

Section: Methodssupporting

confidence: 65%

“…Here, I adapted code previously used to model illumination by visible and near-IR illumination ( Stujenske et al, 2015 ; Podgorski and Ranganathan, 2016 ; Wang et al, 2020 ), simulating illumination and fluorescence emission at visible wavelengths in a ~200 mm 3 volume of mouse grey matter. Spatially detailed models, containing individual tissue elements such as neurons and blood vessels, have proven invaluable for exploring optics with single-neuron resolution ( Charles et al, 2019 ), but require substantial computational resources.…”

Section: Introductionmentioning

confidence: 99%

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Sources of widefield fluorescence from the brain

Waters

2020

eLife

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Widefield fluorescence microscopy is used to monitor the spiking of populations of neurons in the brain. Widefield fluorescence can originate from indicator molecules at all depths in cortex and the relative contributions from somata, dendrites, and axons are often unknown. Here, I simulate widefield illumination and fluorescence collection and determine the main sources of fluorescence for several GCaMP mouse lines. Scattering strongly affects illumination and collection. One consequence is that illumination intensity is greatest ~300–400 µm below the pia, not at the brain surface. Another is that fluorescence from a source deep in cortex may extend across a diameter of 3–4 mm at the brain surface, severely limiting lateral resolution. In many mouse lines, the volume of tissue contributing to fluorescence extends through the full depth of cortex and fluorescence at most surface locations is a weighted average across multiple cortical columns and often more than one cortical area.

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Section: Methodssupporting

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Sources of widefield fluorescence from the brain

Waters

2020

eLife

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“…4). Recent work has shown that for three-photon imaging at ~ 600 µm depth with 1300 nm excitation, heating-induced tissue damage starts at ~ 100 mW average power 34 . Our use of 40-60 mW average power for OGB-1 AM imaging falls within those safety limits.…”

Section: Resultsmentioning

confidence: 99%

Three-photon imaging of synthetic dyes in deep layers of the neocortex

Liu

Roy

Simons

et al. 2020

Sci Rep

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Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~ 450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus in rodents. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration, particularly in non-rodent species. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500–600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5–6 and subsequent three-photon imaging of orientation- and direction- selective visual responses from these cells.

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“…In neurobiology, for instance, coherent optical systems for fluorescence imaging or optogenetic neurostimulation are typically mandated to achieve single-cell resolution targeting. With tissue volumes boasting densities of up to 10 5 neurons/mm 3 and thicknesses of up to 1 mm, such targeting requires dynamic access to several depths at speeds that correspond with the millisecond timescales of neural signalling 1 , 5 , 6 . Similarly, in augmented and virtual reality (AR/VR), accommodating depth cues for 3D images entails the use of axial focusing tools 2 .…”

Section: Introductionmentioning

confidence: 99%

A micromirror array with annular partitioning for high-speed random-access axial focusing

et al. 2020

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Dynamic axial focusing functionality has recently experienced widespread incorporation in microscopy, augmented/virtual reality (AR/VR), adaptive optics and material processing. However, the limitations of existing varifocal tools continue to beset the performance capabilities and operating overhead of the optical systems that mobilize such functionality. The varifocal tools that are the least burdensome to operate (e.g. liquid crystal, elastomeric or optofluidic lenses) suffer from low (≈100 Hz) refresh rates. Conversely, the fastest devices sacrifice either critical capabilities such as their dwelling capacity (e.g. acoustic gradient lenses or monolithic micromechanical mirrors) or low operating overhead (e.g. deformable mirrors). Here, we present a general-purpose random-access axial focusing device that bridges these previously conflicting features of high speed, dwelling capacity and lightweight drive by employing low-rigidity micromirrors that exploit the robustness of defocusing phase profiles. Geometrically, the device consists of an 8.2 mm diameter array of piston-motion and 48-μm-pitch micromirror pixels that provide 2π phase shifting for wavelengths shorter than 1100 nm with 10–90% settling in 64.8 μs (i.e., 15.44 kHz refresh rate). The pixels are electrically partitioned into 32 rings for a driving scheme that enables phase-wrapped operation with circular symmetry and requires <30 V per channel. Optical experiments demonstrated the array’s wide focusing range with a measured ability to target 29 distinct resolvable depth planes. Overall, the features of the proposed array offer the potential for compact, straightforward methods of tackling bottlenecked applications, including high-throughput single-cell targeting in neurobiology and the delivery of dense 3D visual information in AR/VR.

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Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain

Cited by 91 publications

References 51 publications

Sources of widefield fluorescence from the brain

Sources of widefield fluorescence from the brain

Three-photon imaging of synthetic dyes in deep layers of the neocortex

A micromirror array with annular partitioning for high-speed random-access axial focusing

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