scholarly journals High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy

2021 ◽  
Author(s):  
Lina Streich ◽  
Juan Boffi ◽  
Ling Wang ◽  
Khaleel Alhalaseh ◽  
Matteo Barbieri ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Motion Artifacts ◽  
Mammalian Brain ◽  
Neural Cells ◽  
Optical Aberrations ◽  
Photon Excitation ◽  
Tissue Scattering ◽  
Imaging Methodology ◽  
And Function

Multi-photon microscopy has become a powerful tool to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. Yet, tissue scattering, optical aberrations, and motion artifacts degrade the achievable image quality with depth. Here we developed a minimally invasive intravital imaging methodology by combining three-photon excitation, indirect adaptive optics correction, and active electrocardiogram gating to achieve near-diffraction limited resolution up to a depth of 1.2mm in the mouse brain. We demonstrate near-diffraction-limited imaging of deep cortical and sub-cortical dendrites and spines as well as of calcium transients in deep-layer astrocytes in vivo.

scholarly journals High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy

2021 ◽  
Author(s):  
Lina Streich ◽  
Juan Carlos Boffi ◽  
Ling Wang ◽  
Khaleel Alhalaseh ◽  
Matteo Barbieri ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Multiphoton Microscopy ◽  
Motion Artifacts ◽  
Tissue Imaging ◽  
Mammalian Brain ◽  
Neural Cells ◽  
Photon Excitation ◽  
Deep Tissue Imaging ◽  
Fibrous Astrocytes ◽  
And Function

AbstractMultiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. However, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Here we describe a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging. Our modal-based, sensorless AO approach is robust to low signal-to-noise ratios as commonly encountered in deep scattering tissues such as the mouse brain, and permits AO correction over large axial fields of view. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub)cortical dendrites up to a depth of 1.4 mm (the edge of the mouse CA1 hippocampus). In addition, we show applications to deep-layer calcium imaging of astrocytes, including fibrous astrocytes that reside in the highly scattering corpus callosum.

scholarly journals Cross-species Transcriptomic Comparison of In Vitro and In Vivo Mammalian Neural Cells

2015 ◽  
Vol 9 ◽  
pp. BBI.S33124 ◽  
Author(s):  
Peter R. LoVerso ◽  
Christopher M. Wachter ◽  
Feng Cui
Keyword(s):  
Gene Expression ◽  
Transcript Abundance ◽  
Cell Types ◽  
Mammalian Brain ◽  
Neural Cells ◽  
Rna Seq ◽  
Commercial Sources ◽  
And Function

The mammalian brain is characterized by distinct classes of cells that differ in morphology, structure, signaling, and function. Dysregulation of gene expression in these cell populations leads to various neurological disorders. Neural cells often need to be acutely purified from animal brains for research, which requires complicated procedure and specific expertise. Primary culture of these cells in vitro is a viable alternative, but the differences in gene expression of cells grown in vitro and in vivo remain unclear. Here, we cultured three major neural cell classes of rat brain (ie, neurons, astrocytes, and oligodendrocyte precursor cells [OPCs]) obtained from commercial sources. We measured transcript abundance of these cell types by RNA sequencing (RNA-seq) and compared with their counterparts acutely purified from mouse brains. Cross-species RNA-seq data analysis revealed hundreds of genes that are differentially expressed between the cultured and acutely purified cells. Astrocytes have more such genes compared to neurons and OPCs, indicating that signaling pathways are greatly perturbed in cultured astrocytes. This dataset provides a powerful resource to demonstrate the similarities and differences of biological processes in mammalian neural cells grown in vitro and in vivo at the molecular level.

Two-Photon Processor and SeNeCA: a freely available software package to process data from two-photon calcium imaging at speeds down to several milliseconds per frame

2013 ◽  
Vol 110 (1) ◽  
pp. 243-256 ◽  
Author(s):  
Jakub Tomek ◽  
Ondrej Novak ◽  
Josef Syka
Keyword(s):  
Calcium Imaging ◽  
Software Package ◽  
Motion Artifacts ◽  
Calcium Signal ◽  
Neural Cells ◽  
Neuronal System ◽  
Process Data ◽  
Entire Area ◽  
Two Photon

Two-Photon Processor (TPP) is a versatile, ready-to-use, and freely available software package in MATLAB to process data from in vivo two-photon calcium imaging. TPP includes routines to search for cell bodies in full-frame (Search for Neural Cells Accelerated; SeNeCA) and line-scan acquisition, routines for calcium signal calculations, filtering, spike-mining, and routines to construct parametric fields. Searching for somata in artificial in vivo data, our algorithm achieved better performance than human annotators. SeNeCA copes well with uneven background brightness and in-plane motion artifacts, the major problems in simple segmentation methods. In the fast mode, artificial in vivo images with a resolution of 256 × 256 pixels containing ∼100 neurons can be processed at a rate up to 175 frames per second (tested on Intel i7, 8 threads, magnetic hard disk drive). This speed of a segmentation algorithm could bring new possibilities into the field of in vivo optophysiology. With such a short latency (down to 5–6 ms on an ordinary personal computer) and using some contemporary optogenetic tools, it will allow experiments in which a control program can continuously evaluate the occurrence of a particular spatial pattern of activity (a possible correlate of memory or cognition) and subsequently inhibit/stimulate the entire area of the circuit or inhibit/stimulate a different part of the neuronal system. TPP will be freely available on our public web site. Similar all-in-one and freely available software has not yet been published.

scholarly journals Label-free concurrent 5-modal microscopy (Co5M) resolves unknown spatio-temporal processes in wound healing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Markus Seeger ◽  
Christoph Dehner ◽  
Dominik Jüstel ◽  
Vasilis Ntziachristos
Keyword(s):  
Wound Healing ◽  
Label Free ◽  
Third Harmonic ◽  
Two Photon ◽  
Non Invasive ◽  
Photon Excitation ◽  
Free Mode ◽  
Spatio Temporal ◽  
And Function

AbstractThe non-invasive investigation of multiple biological processes remains a methodological challenge as it requires capturing different contrast mechanisms, usually not available with any single modality. Intravital microscopy has played a key role in dynamically studying biological morphology and function, but it is generally limited to resolving a small number of contrasts, typically generated by the use of transgenic labels, disturbing the biological system. We introduce concurrent 5-modal microscopy (Co5M), illustrating a new concept for label-free in vivo observations by simultaneously capturing optoacoustic, two-photon excitation fluorescence, second and third harmonic generation, and brightfield contrast. We apply Co5M to non-invasively visualize multiple wound healing biomarkers and quantitatively monitor a number of processes and features, including longitudinal changes in wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands. Analysis of these parameters offers unique insights into the interplay of wound closure, vasodilation, angiogenesis, skin contracture, and epithelial reformation in space and time, inaccessible by other methods. Co5M challenges the conventional concept of biological observation by yielding multiple simultaneous parameters of pathophysiological processes in a label-free mode.

scholarly journals Epigenetic regulation of neural cell differentiation plasticity in the adult mammalian brain

2008 ◽  
Vol 105 (46) ◽  
pp. 18012-18017 ◽  
Author(s):  
Jun Kohyama ◽  
Takuro Kojima ◽  
Eriko Takatsuka ◽  
Toru Yamashita ◽  
Jun Namiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Mammalian Brain ◽  
Cytokine Signaling ◽  
Specific Gene ◽  
Neural Cells ◽  
Specific Gene Expression

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

scholarly journals Adaptive optics enhanced all-optical photoacoustic tomography

2021 ◽  
Author(s):  
Jakub Czuchnowski ◽  
Robert Prevedel
Keyword(s):  
High Resolution ◽  
Image Quality ◽  
Adaptive Optics ◽  
Photoacoustic Signal ◽  
Photoacoustic Tomography ◽  
Optical Aberrations ◽  
Fabry Perot ◽  
Mammalian Tissues ◽  
All Optical

AbstractAll-optical ultrasound detection bears a number of unique advantages for photoacoustic tomography, including the ability for high resolution sampling of the acoustic field and its compatibility with a wide variety of other optical modalities. However, optical schemes based on miniaturized cavities are sensitive to optical aberrations as well as manufacturing-induced cavity imperfections which degrade sensor sensitivity and deteriorate photoacoustic image quality. Here we present an experimental method based on adaptive optics that is capable of enhancing the overall sensitivity of Fabry-Pérot based photoacoustic sensors. We experimentally observe clear improvements in photoacoustic signal detection as well as overall image quality after photoacoustic tomography reconstructions when applied to mammalian tissues in vivo.

scholarly journals Dynamic super-resolution structured illumination imaging in the living brain

2019 ◽  
Vol 116 (19) ◽  
pp. 9586-9591 ◽  
Author(s):  
Raphaël Turcotte ◽  
Yajie Liang ◽  
Masashi Tanimoto ◽  
Qinrong Zhang ◽  
Ziwei Li ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Optical Aberrations ◽  
Sample Motion ◽  
Heterogeneous Tissue ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

scholarly journals Fast widefield imaging of neuronal structure and function with optical sectioning in vivo

2020 ◽  
Vol 6 (19) ◽  
pp. eaaz3870 ◽  
Author(s):  
Ziwei Li ◽  
Qinrong Zhang ◽  
Shih-Wei Chou ◽  
Zachary Newman ◽  
Raphaël Turcotte ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
In Vivo Imaging ◽  
Neuromuscular Junctions ◽  
Structure And Function ◽  
Structured Illumination ◽  
Neuronal Structure ◽  
Optical Sectioning ◽  
Structured Illumination Microscopy ◽  
And Function

Optical microscopy, owing to its noninvasiveness and subcellular resolution, enables in vivo visualization of neuronal structure and function in the physiological context. Optical-sectioning structured illumination microscopy (OS-SIM) is a widefield fluorescence imaging technique that uses structured illumination patterns to encode in-focus structures and optically sections 3D samples. However, its application to in vivo imaging has been limited. In this study, we optimized OS-SIM for in vivo neural imaging. We modified OS-SIM reconstruction algorithms to improve signal-to-noise ratio and correct motion-induced artifacts in live samples. Incorporating an adaptive optics (AO) module to OS-SIM, we found that correcting sample-induced optical aberrations was essential for achieving accurate structural and functional characterizations in vivo. With AO OS-SIM, we demonstrated fast, high-resolution in vivo imaging with optical sectioning for structural imaging of mouse cortical neurons and zebrafish larval motor neurons, and functional imaging of quantal synaptic transmission at Drosophila larval neuromuscular junctions.

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