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1.
Cell ; 186(11): 2475-2491.e22, 2023 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-37178688

RESUMEN

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.


Asunto(s)
Imagenología Tridimensional , Animales , Ratones , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos
2.
Cell ; 184(12): 3318-3332.e17, 2021 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-34038702

RESUMEN

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.


Asunto(s)
Algoritmos , Imagenología Tridimensional , Óptica y Fotónica , Tomografía , Animales , Calcio/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Drosophila , Células HeLa , Humanos , Larva/fisiología , Hígado/diagnóstico por imagen , Masculino , Ratones Endogámicos C57BL , Neoplasias/patología , Ratas Sprague-Dawley , Relación Señal-Ruido , Fracciones Subcelulares/fisiología , Factores de Tiempo , Pez Cebra
3.
Neurophotonics ; 9(4): 041407, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-35450225

RESUMEN

Significance: Mesoscale neural imaging in vivo has gained extreme popularity in neuroscience for its capacity of recording large-scale neurons in action. Optical imaging with single-cell resolution and millimeter-level field of view in vivo has been providing an accumulated database of neuron-behavior correspondence. Meanwhile, optical detection of neuron signals is easily contaminated by noises, background, crosstalk, and motion artifacts, while neural-level signal processing and network-level coordinate are extremely complicated, leading to laborious and challenging signal processing demands. The existing data analysis procedure remains unstandardized, which could be daunting to neophytes or neuroscientists without computational background. Aim: We hope to provide a general data analysis pipeline of mesoscale neural imaging shared between imaging modalities and systems. Approach: We divide the pipeline into two main stages. The first stage focuses on extracting high-fidelity neural responses at single-cell level from raw images, including motion registration, image denoising, neuron segmentation, and signal extraction. The second stage focuses on data mining, including neural functional mapping, clustering, and brain-wide network deduction. Results: Here, we introduce the general pipeline of processing the mesoscale neural images. We explain the principles of these procedures and compare different approaches and their application scopes with detailed discussions about the shortcomings and remaining challenges. Conclusions: There are great challenges and opportunities brought by the large-scale mesoscale data, such as the balance between fidelity and efficiency, increasing computational load, and neural network interpretability. We believe that global circuits on single-neuron level will be more extensively explored in the future.

4.
Nat Protoc ; 17(9): 1953-1979, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35768724

RESUMEN

With the development of a wide variety of animal models in recent years, there is a rapidly growing demand for long-term, high-speed intravital fluorescence imaging to observe intercellular and intracellular interactions in their native states. Scanning light-field microscopy (sLFM) with digital adaptive optics provides a compact computational solution by imaging the entire volume in a tomographic way with orders-of-magnitude improvement in spatiotemporal resolution and reduction in phototoxicity, as compared to traditional intravital microscopy. Here, we present a step-by-step protocol for both hardware and software implementation of multicolor sLFM as an add-on to a normal wide-field fluorescence microscope by using off-the-shelf lenses and devices at low cost. The procedure can be easily applied to other LFM variants, which can be advantageous in certain experimental contexts. Owing to the strong reliance of sLFM on algorithmic post-processing for high-quality data, the protocol describes various kinds of artefacts and corresponding parameters used for correcting and performance optimization. To increase the tolerance to system misalignment and differences in device fabrication, we describe a one-step calibration method for robust imaging performance up to the diffraction limit. An open-source graphical user interface is presented for hardware synchronization and real-time rendering of multiview images. The whole procedure including optical setup, software installation, system calibration and 3D reconstruction can be executed in 3-4 d with basic knowledge in optics and electronics.


Asunto(s)
Lentes , Microscopía , Animales , Calibración , Microscopía/métodos , Óptica y Fotónica , Programas Informáticos
5.
Front Hum Neurosci ; 15: 701923, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35002649

RESUMEN

Previous studies used BA and BEI structures as stimuli to infer that syntax-first models seemed not applicable in Chinese. However, there were inconsistent results of both within same structures and between different structures. Since sentence structures of stimuli were non-canonical as well as lacking wide representativeness in Chinese, we examined the processing mechanism of a more representative structure in Chinese, QING (QING + NP1 + V + NP2) structure in the current study. Four conditions, including correct sentences (CORRECT), semantic-violated sentences (SEMANTIC), syntactic-violated sentences (SYNTACTIC), and combined violated sentences (COMBINED), were composed by manipulating the V between NP1 and NP2. Results with respect to three types of violation were as follows. In the initial phrase (100-300 ms), there existed an interaction between SEMANTIC consistency and the SYNTACTIC category. In the intermediate phrase (300-500 ms), the interaction continued with similar negative waves evoked by three types of violated sentences. In the final phrase (500-700 ms), both SYNTACTIC or COMBINED evoked obvious negative waves. The current research of Qing structure provided new evidence for the processing mechanism of Chinese sentence patterns. Specifically, we found that the interactive model rather than the syntax-first model may apply to the processing of this specific structure of Chinese sentences and compared the results with those reported in previous studies that examined other types of sentence structures.

6.
Nat Commun ; 12(1): 6391, 2021 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-34737278

RESUMEN

Quantitative volumetric fluorescence imaging at high speed across a long term is vital to understand various cellular and subcellular behaviors in living organisms. Light-field microscopy provides a compact computational solution by imaging the entire volume in a tomographic way, while facing severe degradation in scattering tissue or densely-labelled samples. To address this problem, we propose an incoherent multiscale scattering model in a complete space for quantitative 3D reconstruction in complicated environments, which is called computational optical sectioning. Without the requirement of any hardware modifications, our method can be generally applied to different light-field schemes with reduction in background fluorescence, reconstruction artifacts, and computational costs, facilitating more practical applications of LFM in a broad community. We validate the superior performance by imaging various biological dynamics in Drosophila embryos, zebrafish larvae, and mice.


Asunto(s)
Microscopía Fluorescente/métodos , Animales , Embrión no Mamífero/diagnóstico por imagen , Imagenología Tridimensional/métodos , Larva , Ratones , Pez Cebra
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