scholarly journals Cortical Column and Whole Brain Imaging of Neural Circuits with Molecular Contrast and Nanoscale Resolution

2018 ◽  
Author(s):  
Ruixuan Gao ◽  
Shoh M. Asano ◽  
Srigokul Upadhyayula ◽  
Pisarev Igor ◽  
Daniel E. Milkie ◽  
...  
Keyword(s):  
Neural Development ◽  
High Speed ◽  
Large Scale ◽  
Neural Circuits ◽  
Light Sheet ◽  
Synaptic Proteins ◽  
Light Sheet Microscopy ◽  
Mouse Cortex ◽  
Molecular Contrast ◽  
The Brain

AbstractOptical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.One Sentence SummaryCombined expansion and lattice light sheet microscopy enables high speed, nanoscale molecular imaging of neural circuits over large volumes.

scholarly journals Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution

Science ◽  
2019 ◽  
Vol 363 (6424) ◽  
pp. eaau8302 ◽  
Author(s):  
Ruixuan Gao ◽  
Shoh M. Asano ◽  
Srigokul Upadhyayula ◽  
Igor Pisarev ◽  
Daniel E. Milkie ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Neural Development ◽  
Dopaminergic Neurons ◽  
Large Scale ◽  
Light Sheet ◽  
Synaptic Proteins ◽  
Light Sheet Microscopy ◽  
Mouse Cortex ◽  
Molecular Contrast ◽  
The Brain

Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entireDrosophilabrain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

scholarly journals Scalable volumetric imaging for ultrahigh-speed brain mapping at synaptic resolution

2017 ◽  
Author(s):  
Hao Wang ◽  
Qingyuan Zhu ◽  
Lufeng Ding ◽  
Yan Shen ◽  
Chao-Yu Yang ◽  
...  
Keyword(s):  
Brain Mapping ◽  
High Speed ◽  
Large Scale ◽  
Light Sheet ◽  
Volumetric Imaging ◽  
Tissue Sections ◽  
Light Sheet Microscopy ◽  
Cell Type Specific ◽  
Microscopy Method ◽  
Immunofluorescence Labeling

We describe a new light-sheet microscopy method for fast, large-scale volumetric imaging. Combining synchronized scanning illumination and oblique imaging over cleared, thick tissue sections in smooth motion, our approach achieves high-speed 3D image acquisition of an entire mouse brain within 2 hours, at a resolution capable of resolving synaptic spines. It is compatible with immunofluorescence labeling, enabling flexible cell-type specific brain mapping, and is readily scalable for large biological samples such as primate brain.

scholarly journals Rapid reconstruction of neural circuits using tissue expansion and lattice light sheet microscopy

2021 ◽  
Author(s):  
Joshua L Lillvis ◽  
Hideo Otsuna ◽  
Xiaoyu Ding ◽  
Igor Pisarev ◽  
Takashi Kawase ◽  
...  
Keyword(s):  
Neural Circuits ◽  
Structural Connectivity ◽  
Tissue Expansion ◽  
Light Sheet ◽  
Structure And Function ◽  
Analysis Pipeline ◽  
Light Sheet Microscopy ◽  
Reference Specimens ◽  
Molecular Contrast ◽  
And Function

Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

scholarly journals High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics

2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Benjamin Schmid ◽  
Gopi Shah ◽  
Nico Scherf ◽  
Michael Weber ◽  
Konstantin Thierbach ◽  
...  
Keyword(s):  
High Speed ◽  
Light Sheet ◽  
Endodermal Cell ◽  
Cell Dynamics ◽  
Light Sheet Microscopy

Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

2012 ◽  
Vol 9 (7) ◽  
pp. 755-763 ◽  
Author(s):  
Raju Tomer ◽  
Khaled Khairy ◽  
Fernando Amat ◽  
Philipp J Keller
Keyword(s):  
High Speed ◽  
Light Sheet ◽  
High Speed Imaging ◽  
Light Sheet Microscopy

scholarly journals Imaging the dynamic recruitment of monocytes to the blood–brain barrier and specific brain regions during Toxoplasma gondii infection

2019 ◽  
Vol 116 (49) ◽  
pp. 24796-24807 ◽  
Author(s):  
Christine A. Schneider ◽  
Dario X. Figueroa Velez ◽  
Ricardo Azevedo ◽  
Evelyn M. Hoover ◽  
Cuong J. Tran ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Blood Brain Barrier ◽  
Light Sheet ◽  
Brain Regions ◽  
Brain Barrier ◽  
Cell Segmentation ◽  
Cns Infection ◽  
Light Sheet Microscopy ◽  
Blood Brain ◽  
The Brain

Brain infection by the parasite Toxoplasma gondii in mice is thought to generate vulnerability to predation by mechanisms that remain elusive. Monocytes play a key role in host defense and inflammation and are critical for controlling T. gondii. However, the dynamic and regional relationship between brain-infiltrating monocytes and parasites is unknown. We report the mobilization of inflammatory (CCR2+Ly6Chi) and patrolling (CX3CR1+Ly6Clo) monocytes into the blood and brain during T. gondii infection of C57BL/6J and CCR2RFP/+CX3CR1GFP/+ mice. Longitudinal analysis of mice using 2-photon intravital imaging of the brain through cranial windows revealed that CCR2-RFP monocytes were recruited to the blood–brain barrier (BBB) within 2 wk of T. gondii infection, exhibited distinct rolling and crawling behavior, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact T. gondii-infected brains using iDISCO+ and light-sheet microscopy enabled global 3D detection of monocytes. Clusters of T. gondii and individual monocytes across the brain were identified using an automated cell segmentation pipeline, and monocytes were found to be significantly correlated with sites of T. gondii clusters. Computational alignment of brains to the Allen annotated reference atlas [E. S. Lein et al., Nature 445:168–176 (2007)] indicated a consistent pattern of monocyte infiltration during T. gondii infection to the olfactory tubercle, in contrast to LPS treatment of mice, which resulted in a diffuse distribution of monocytes across multiple brain regions. These data provide insights into the dynamics of monocyte recruitment to the BBB and the highly regionalized localization of monocytes in the brain during T. gondii CNS infection.

scholarly journals Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

2020 ◽  
Vol 14 ◽  
Author(s):  
Bo M. B. Bekkouche ◽  
Helena K. M. Fritz ◽  
Elisa Rigosi ◽  
David C. O'Carroll
Keyword(s):  
Refractive Index ◽  
Rapid Development ◽  
Vacuum Treatment ◽  
Building Blocks ◽  
Light Sheet ◽  
Tracheal System ◽  
Light Sheet Microscopy ◽  
Tissue Shrinkage ◽  
Tunable Refractive Index ◽  
The Brain

Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (“MS/P”) with several more recently applied clearing media that offer tunable refractive index (n): 2,2′-thiodiethanol (TDE), “SeeDB2” (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

Efficient processing and analysis of large-scale light-sheet microscopy data

2015 ◽  
Vol 10 (11) ◽  
pp. 1679-1696 ◽  
Author(s):  
Fernando Amat ◽  
Burkhard Höckendorf ◽  
Yinan Wan ◽  
William C Lemon ◽  
Katie McDole ◽  
...  
Keyword(s):  
Large Scale ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Efficient Processing ◽  
Microscopy Data

scholarly journals Reflective imaging improves resolution, speed, and collection efficiency in light sheet microscopy

2017 ◽  
Author(s):  
Yicong Wu ◽  
Abhishek Kumar ◽  
Corey Smith ◽  
Evan Ardiel ◽  
Panagiotis Chandris ◽  
...  
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Collection Efficiency ◽  
Single Cells ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Three Dimensions ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Simultaneous Acquisition

AbstractLight-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 μm) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

scholarly journals Large-scale voltage imaging in the brain using targeted illumination

2021 ◽  
Author(s):  
Sheng Xiao ◽  
Eric Lowet ◽  
Howard Gritton ◽  
Pierre Fabris ◽  
Yangyang Wang ◽  
...  
Keyword(s):  
High Speed ◽  
Large Scale ◽  
Low Cost ◽  
Mammalian Brain ◽  
Voltage Imaging ◽  
Background Rejection ◽  
Large Scale Network ◽  
Voltage Signal ◽  
Signal Contrast ◽  
The Brain

Recent improvements in genetically encoded voltage indicators enabled high precision imaging of single neuron's action potentials and subthreshold membrane voltage dynamics in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool to record activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy prone to background signal contamination. We implemented a simple, low cost, targeted illumination strategy based on a digital micromirror device (DMD) to restrict illumination to the cells of interest to improve background rejection, and quantified optical voltage signal improvement in neurons expressing the fully genetically encoded voltage indicator SomArchon. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brains of awake mice. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over several minutes in the brain. Thus, the DMD-based targeted illumination strategy described here offers a simple solution for high-speed voltage imaging analysis of large scale network at the millisecond time scale with single cell resolution in the brains of behaving animals.

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