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.

scholarly journals Comparison of transparency and shrinkage of insect brains using novel and traditional clearing methods

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

AbstractImprovement 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 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 & SeeDB2G matched to oil and glycerol immersion, n=1.52 & 1.47 respectively) and Rapiclear (also with n=1.52 & 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, these newly 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.

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 Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Duncan P. Ryan ◽  
Elizabeth A. Gould ◽  
Gregory J. Seedorf ◽  
Omid Masihzadeh ◽  
Steven H. Abman ◽  
...  
Keyword(s):  
Refractive Index ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals 3D Reconstruction of the Clarified Rat Hindbrain Choroid Plexus

2021 ◽  
Vol 9 ◽  
Author(s):  
Paola Perin ◽  
Riccardo Rossetti ◽  
Carolina Ricci ◽  
Daniele Cossellu ◽  
Simone Lazzarini ◽  
...  
Keyword(s):  
Choroid Plexus ◽  
Spatial Organization ◽  
Light Sheet ◽  
Dorsal Cochlear Nucleus ◽  
Horizontal Segment ◽  
Venous Outflow ◽  
Light Sheet Microscopy ◽  
Contact Points ◽  
The Brain ◽  
4Th Ventricle

The choroid plexus (CP) acts as a regulated gate between blood and cerebrospinal fluid (CSF). Despite its simple histology (a monostratified cuboidal epithelium overlying a vascularized stroma), this organ has remarkably complex functions several of which involve local interaction with cells located around ventricle walls. Our knowledge of CP structural organization is mainly derived from resin casts, which capture the overall features but only allow reconstruction of the vascular pattern surface, unrelated to the overlying epithelium and only loosely related to ventricular location. Recently, CP single cell atlases are starting to emerge, providing insight on local heterogeneities and interactions. So far, however, few studies have described CP spatial organization at the mesoscale level, because of its fragile nature and deep location within the brain. Here, using an iDISCO-based clearing approach and light-sheet microscopy, we have reconstructed the normal rat hindbrain CP (hCP) macro- and microstructure, using markers for epithelium, arteries, microvasculature, and macrophages, and noted its association with 4th ventricle-related neurovascular structures. The hCP is organized in domains associated to a main vessel (fronds) which carry a variable number of villi; the latter are enclosed by epithelium and may be flat (leaf-like) or rolled up to variable extent. Arteries feeding the hCP emerge from the cerebellar surface, and branch into straight arterioles terminating as small capillary anastomotic networks, which run within a single villus and terminate attaching multiple times to a large tortuous capillary (LTC) which ends into a vein. Venous outflow mostly follows arterial pathways, except for the lateral horizontal segment (LHS) and the caudal sagittal segment. The structure of fronds and villi is related to the microvascular pattern at the hCP surface: when LTCs predominate, leaflike villi are more evident and bulge from the surface; different, corkscrew-like villi are observed in association to arterioles reaching close to the CP surface with spiraling capillaries surrounding them. Both leaf-like and corkscrew-like villi may reach the 4th ventricle floor, making contact points at their tip, where no gap is seen between CP epithelium and ependyma. Contacts usually involve several adjacent villi and may harbor epiplexus macrophages. At the junction between medial (MHS) and lateral (LHS) horizontal segment, arterial supply is connected to the temporal bone subarcuate fossa, and venous outflow drains to a ventral vein which exits through the cochlear nuclei at the Luschka foramen. These vascular connections stabilize the hCP overall structure within the 4th ventricle but make MHS-LHS joint particularly fragile and very easily damaged when removing the brain from the skull. Even in damaged samples, however, CP fronds (or isolated villi) often remain strongly attached to the dorsal cochlear nucleus (DCN) surface; in these fronds, contacts are still present and connecting “bridges” may be seen, suggesting the presence of real molecular contacts rather than mere appositions.

scholarly journals Glymphatic pathways in the gyrencephalic brain

2021 ◽  
pp. 0271678X2199617
Author(s):  
Nicholas Burdon Bèchet ◽  
Nagesh C Shanbhag ◽  
Iben Lundgaard
Keyword(s):  
Interstitial Fluid ◽  
Three Dimensional ◽  
Light Sheet ◽  
Subcortical Structures ◽  
Large Mammal ◽  
Glymphatic System ◽  
Light Sheet Microscopy ◽  
Tracer Distribution ◽  
The Brain ◽  
Solute Clearance

Identification of the perivascular compartment as the point of exchange between cerebrospinal fluid (CSF) and interstitial fluid mediating solute clearance in the brain, named the glymphatic system, has emerged as an important clearance pathway for neurotoxic peptides such as amyloid-beta. However, the foundational science of the glymphatic system is based on rodent studies. Here we investigated whether the glymphatic system exists in a large mammal with a highly gyrified brain. CSF penetration into the brain via perivascular pathways, a hallmark of glymphatic function, was seen throughout the gyrencephalic cortex and subcortical structures, validating the conservation of the glymphatic system in a large mammal. Macroscopic CSF tracer distribution followed the sulci and fissures showing that these folds enhance CSF dispersion. Three-dimensional renditions from light sheet microscopy showed a PVS influx density 4-fold larger in the pig brain than in mice. This demonstrates the existence of an advanced solute transport system in the gyrencephalic brain that could be utilised therapeutically for enhancing waste clearance.

scholarly journals Acoustic light-sheet microscopy

2021 ◽  
Author(s):  
Stefan Wunderl ◽  
Ayumu Ishijima ◽  
Etsuo Susaki ◽  
Zihui Xu ◽  
Hong Song ◽  
...  
Keyword(s):  
Light Propagation ◽  
High Spatial Resolution ◽  
Single Cells ◽  
Acoustic Pulse ◽  
Light Sheet ◽  
Wide Field ◽  
Axial Resolution ◽  
3D Objects ◽  
Light Sheet Microscopy ◽  
The Brain

Light-sheet imaging of 3D objects with high spatial resolution remains an open challenge because of the trade-off between field-of-view (FOV) and axial resolution originating from the diffraction of light. We developed acoustic light-sheet microscopy (acoustic LSM), which actively manipulates the light propagation inside a large sample to obtain wide-field microscopic images deep inside a target. By accurately coupling a light-sheet illumination pulse into a planar acoustic pulse, the light-sheet can be continuously guided over large distances. We imaged a fluorescence-labeled transparent mouse brain for the FOVs of 19.3 x 12.4 mm2 and 9.7 x 5.9 mm2 with resolved microstructures and single cells deep inside the brain. Acoustic LSM creates new opportunities for the application of light-sheet in the field of industry to basic science.

scholarly journals DSS-induced inflammation in the colon drives a pro-inflammatory signature in the brain that is ameliorated by prophylactic treatment with the S100A9 inhibitor paquinimod

2021 ◽  
Author(s):  
Sarah Talley ◽  
Rasa Valiauga ◽  
Lillian Anderson ◽  
Abigail R Cannon ◽  
Mashkoor A Choudhry ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Inflammatory Cytokines ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Light Sheet ◽  
Positive Control ◽  
Light Sheet Microscopy ◽  
Cytokines And Chemokines ◽  
Organ Systems ◽  
The Brain

Background: Inflammatory Bowel Disease (IBD) is established to drive pathological sequelae in organ systems outside the intestine, including the central nervous system (CNS). Many patients exhibit cognitive deficits, particularly during disease flare. The connection between colonic inflammation and neuroinflammation remains unclear and characterization of the neuroinflammatory phenotype in the brain during colitis is ill-defined. Methods: Transgenic mice expressing a bioluminescent reporter of active caspase-1 were treated with 2% Dextran Sodium Sulfate (DSS) for 7 days to induce acute colitis, and colonic, systemic and neuroinflammation were assessed. In some experiments, mice were prophylactically treated with paquinimod (ABR-215757) to inhibit S100A9 inflammatory signaling. As a positive control for peripheral-induced neuroinflammation, mice were injected with lipopolysaccharide (LPS). Colonic, systemic and brain inflammatory cytokines and chemokines were measured by cytokine bead array (CBA) and Proteome profiler mouse cytokine array. Bioluminescence was quantified in the brain and caspase activation was confirmed by immunoblot. Immune cell infiltration into the CNS was measured by flow cytometry, while light sheet microscopy was used to monitor changes in resident microglia localization in intact brains during DSS or LPS-induced neuroinflammation. RNA sequencing was performed to identify transcriptomic changes occurring in the CNS of DSS-treated mice. Expression of inflammatory biomarkers were quantified in the brain and serum by qRT-PCR, ELISA and WB. Results: DSS-treated mice exhibited clinical hallmarks of colitis, including weight loss, colonic shortening and inflammation in the colon. We also detected a significant increase in inflammatory cytokines in the serum and brain, as well as caspase and microglia activation in the brain of mice with ongoing colitis. RNA sequencing of brains isolated from DSS-treated mice revealed differential expression of genes involved in the regulation of inflammatory responses. This inflammatory phenotype was similar to the signature detected in LPS-treated mice, albeit less robust and transient, as inflammatory gene expression returned to baseline following cessation of DSS. Pharmacological inhibition of S100A9, one of the transcripts identified by RNA sequencing, attenuated colitis severity and systemic and neuroinflammation. Conclusions: Our findings suggest that local inflammation in the colon drives systemic inflammation and neuroinflammation, and this can be ameliorated by inhibition of the S100 alarmin, S100A9.

scholarly journals Three-dimensional, isotropic imaging of mouse brain using multi-view deconvolution light sheet microscopy

2017 ◽  
Vol 10 (05) ◽  
pp. 1743006 ◽  
Author(s):  
Sa Liu ◽  
Jun Nie ◽  
Yusha Li ◽  
Tingting Yu ◽  
Dan Zhu ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Pyramidal Neurons ◽  
Structural Information ◽  
Fluorescent Microscopy ◽  
Three Dimensional ◽  
Light Sheet ◽  
Nerve Fibers ◽  
Single View ◽  
Light Sheet Microscopy ◽  
The Brain

We present a three-dimensional (3D) isotropic imaging of mouse brain using light-sheet fluorescent microscopy (LSFM) in conjunction with a multi-view imaging computation. Unlike common single view LSFM is used for mouse brain imaging, the brain tissue is 3D imaged under eight views in our study, by a home-built selective plane illumination microscopy (SPIM). An output image containing complete structural information as well as significantly improved resolution ([Formula: see text]4 times) are then computed based on these eight views of data, using a bead-guided multi-view registration and deconvolution. With superior imaging quality, the astrocyte and pyramidal neurons together with their subcellular nerve fibers can be clearly visualized and segmented. With further including other computational methods, this study can be potentially scaled up to map the connectome of whole mouse brain with a simple light-sheet microscope.

Exploring the brain on multiple scales with correlative two-photon and light sheet microscopy

2014 ◽  
Author(s):  
Ludovico Silvestri ◽  
Anna Letizia Allegra Mascaro ◽  
Irene Costantini ◽  
Leonardo Sacconi ◽  
Francesco S. Pavone
Keyword(s):  
Multiple Scales ◽  
Light Sheet ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
The Brain

scholarly journals Fast volumetric mapping of human brain slices

2020 ◽  
Author(s):  
Luca Pesce ◽  
Annunziatina Laurino ◽  
Vladislav Gavryusev ◽  
Giacomo Mazzamuto ◽  
Giuseppe Sancataldo ◽  
...  
Keyword(s):  
Human Brain ◽  
Brain Connectivity ◽  
Brain Slices ◽  
Light Sheet ◽  
Volumetric Imaging ◽  
Neuronal Markers ◽  
Light Sheet Microscopy ◽  
Inhibitory Population ◽  
Anatomical Information ◽  
The Brain

AbstractWe still lack a detailed map of the anatomical disposition of neurons in the human brain. A complete map would be an important step for deeply understanding the brain function, providing anatomical information useful to decipher the neuronal pattern in healthy and diseased conditions. Here, we present several important advances towards this goal, obtained by combining a new clearing method, advanced Light Sheet Microscopy and automated machinelearning based image analysis. We perform volumetric imaging of large sequentially stained human brain slices, labelled for two different neuronal markers NeuN and GAD67, discriminating the inhibitory population and reconstructing the brain connectivity.

Sign in / Sign up

Export Citation Format

Share Document