Scholler J, Jonsson J, Jordá-Siquier T, Gantar I, Batti L, Cheeseman BL, Pagès S, Sbalzarini IF, Lamy CM. Efficient image analysis for large-scale next generation histopathology using pAPRica. bioRxiv [Preprint] 2023: 2023.01.27.525687. doi: 10.1101/2023.01.27.525687.

 

Background: Next-generation histopathology enables volumetric imaging of entire organs at high resolution, but the resulting datasets – often reaching terabyte or even petabyte scales – pose major challenges for data processing and analysis. Current methods require high-performance computing infrastructure and are too slow for clinical use or high-throughput research.

 

Hypothesis: This study tested the hypothesis that an Adaptive Particle Representation (APR)-based image analysis pipeline can efficiently process large-scale, multi-channel histological datasets acquired with light-sheet microscopy – specifically ClearScope – on standard workstations, while maintaining analytical accuracy.

 

Methods: The authors developed pAPRica, an APR-native processing framework that performs stitching, segmentation, registration and visualization directly on particle-based representations. A key test involved imaging a 1.7×2.7×0.3 cm³ human brain tissue block from an Alzheimer’s disease case using a ClearScope. The microscope employed dual digitally-scanned light sheets and chromatic aberration correction to capture high-resolution, multi-channel data (3.9 TB total). Each tile was converted to APR format in real time, allowing efficient segmentation and alignment during acquisition.

 

Results: APR conversion enabled a 117-fold reduction in data size and a 66-fold increase in processing speed relative to voxel-based methods. Segmentation of Aβ plaques achieved >99.9% object-matching accuracy compared to voxel-based ground truth. The framework also enabled cortical depth and density analysis of plaques. Performance was consistent across synthetic petascale datasets.

 

Conclusions: The integration of ClearScope imaging with APR-native analysis through pAPRica provides a scalable and accurate solution for real-time processing of large 3D histological datasets. This approach supports whole-organ analysis and has direct applicability in both clinical diagnostics and large-scale research projects.

Dayton H, Kiss J, Wei M, Chauhan S, LaMarre E, Cornell WC, Morgan CJ, Janakiraman A, Min W, Tomer R, Price-Whelan A, Nirody JA, Dietrich LEP. Cellular arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms. PLoS Biol 2024;22(2):e3002205. doi: 10.1371/journal.pbio.3002205.

 

Background: Biofilms are structured bacterial communities where access to nutrients and antibiotics is limited. In Pseudomonas aeruginosa, spatial cell arrangement may influence metabolic activity and drug tolerance, but the mechanistic links between microanatomy and physiology remain poorly understood.

 

Hypothesis: This study tested the hypothesis that the physical organization of cells within P. aeruginosa biofilms governs resource distribution, metabolic activity and antibiotic susceptibility.

 

Methods: The authors used a ClearScope to visualize clonal cell arrangement across biofilm depth. A targeted screen of 48 mutants disrupted in biofilm-related genes was conducted to identify factors affecting spatial organization. To evaluate physiological consequences, stimulated Raman scattering (SRS) microscopy was separately employed to map metabolic activity via deuterium incorporation. Substrate penetration and antibiotic response were assessed using fluorescent tracers, inducible reporters and propidium iodide staining after tobramycin exposure.

 

Results: Wild-type biofilms formed vertically aligned striations of clonal cells that corresponded with peak metabolic activity. Mutants lacking functional pili or O-antigen structures displayed disordered arrangements, altered nutrient penetration and shifts in metabolic zones. These changes increased susceptibility to tobramycin near the biofilm surface.

 

Conclusions: Biofilm microanatomy, governed by genetic determinants such as pili and O-antigen structures, modulates internal resource distribution and drug susceptibility. These findings highlight spatial organization as a key determinant of biofilm physiology and suggest novel strategies for improving antimicrobial efficacy.

Datta MS, Chen Y, Chauhan S, Zhang J, De La Cruz ED, Gong C, Tomer R. Whole-brain mapping reveals the divergent impact of ketamine on the dopamine system. Cell Rep 2023;42(12):113491. doi: 10.1016/j.celrep.2023.113491.

 

Background: Ketamine, a clinically relevant anesthetic and fast-acting antidepressant, has complex effects on the brain’s neurotransmitter systems. While acute ketamine effects on dopamine (DA) activity are well characterized, the long-term, brain-wide impact of chronic ketamine exposure remains poorly understood.

 

Hypothesis: This study tested the hypothesis that repeated ketamine exposure leads to dose-dependent and region-specific structural plasticity in the brain’s dopaminergic system.

 

Methods: The authors developed a high-resolution whole-brain phenotyping platform incorporating tissue clearing, immunolabeling and ClearScope light-sheet theta microscopy to map sub-cellular dopaminergic changes in male mice following chronic ketamine administration. The imaging was integrated with “suiteWB,” a computational pipeline enabling quantitative analysis of tyrosine hydroxylase positive (TH+) neuron counts and projections across annotated brain regions.

 

Results: Ten days of repeated ketamine exposure (30 or 100 mg/kg) resulted in divergent, dose-dependent alterations in TH+ neurons. Decreases were observed in midbrain regions linked to behavioral states (e.g., dorsal raphe, retrorubral area), while increases occurred in hypothalamic domains (e.g., arcuate nucleus, zona incerta). Additionally, projection density analyses revealed enhanced TH+ innervation of associative cortices (e.g., prefrontal cortex) and reduced projections to sensory and striatal regions. The study also identified previously untranslated TH mRNA+ neurons as a potential substrate for ketamine-induced plasticity, implicating post-transcriptional regulation.

 

Conclusions: Chronic ketamine induces complex and region-specific remodeling of the dopaminergic system, involving both gain and loss of TH+ neurons and their projections. These findings underscore the importance of unbiased, whole-brain analyses to understand the structural basis of ketamine’s therapeutic and adverse effects.

Fay MG, Lang PJ, Denu DS, O’Connor NJ, Haydock B, Blaisdell J, Roussel N, Wilson A, Aronson SM, Angstman PJ, Gong C, Butola T, Devinsky O, Basu J, Tomer R, Glaser JR. ClearScope: a fully integrated light sheet theta microscope for sub-cellular resolution imaging without lateral size constraints. bioRxiv [Preprint]. 2024:2024.08.15.608141. doi: 10.1101/2024.08.15.608141.

 

Background: Three-dimensional (3D) imaging of cleared, intact brain tissue is essential for mapping neural connectivity and investigating structural changes in neurological and neuropsychiatric disorders. Light-sheet fluorescence microscopy (LSFM) offers rapid, high-resolution imaging but is limited by lateral (XY) specimen size constraints due to its orthogonal optical design.

 

Hypothesis: This study tested the hypothesis that a fully integrated light sheet theta microscopy (LSTM) system – ClearScope – can achieve sub-cellular resolution imaging of intact, cleared specimens without lateral size limitations, while providing an accessible and streamlined workflow.

 

Methods: ClearScope employs dual oblique illumination and a perpendicular detection path, enabling deep tissue imaging regardless of lateral dimensions. It integrates an automated detection objective changer, adaptive refractive index mismatch correction, intelligent tissue scanning workflows and comprehensive pre- and post-processing tools. Performance was evaluated using fluorescent microspheres and biological samples, including whole mouse brains and human cortex.

 

Results: ClearScope achieves lateral resolution of ~0.33 µm using a 20x detection objective and axial resolutions of 5.4–6.7 µm across the field of view. Imaging intact mouse brains and human tissue yielded high-quality, minimally distorted 3D images suitable for digital neuron tracing, vascular reconstruction and stereologic analysis. The system reliably handles large samples and varied refractive indices with intuitive user interfaces.

https://pubmed.ncbi.nlm.nih.gov/39229056/

 

Conclusions: ClearScope advances volumetric imaging by enabling sub-cellular resolution without lateral size constraints. Its integrated, user-friendly platform expands access to whole-organ imaging in neuroscience, systems biology and related disciplines.

 

 

Migliori B, Datta MS, Dupre C, Apak MC, Asano S, Gao R, Boyden ES, Hermanson O, Yuste R, Tomer R. Light sheet theta microscopy for rapid high-resolution imaging of large biological samples. BMC Biol 2018;16(1):57. doi: 10.1186/s12915-018-0521-8.

 

Background: Light sheet fluorescence microscopy (LSFM) has advanced large-scale volumetric imaging in biology, particularly in cleared tissue samples. However, conventional LSFM configurations often face trade-offs between axial resolution, field of view and working distance, especially when imaging large intact organs like mouse brains.

 

Hypothesis: This study tested the hypothesis that reconfiguring the light sheet illumination geometry can overcome resolution and coverage limitations in conventional LSFM, enabling uniform high-resolution imaging across large, intact biological samples.

 

Methods: The authors developed Light Sheet Theta Microscopy (LSTM), a novel LSFM architecture in which two obliquely oriented illumination arms deliver light sheets at a non-orthogonal (theta) angle to the detection objective. Unlike conventional orthogonal LSFM, LSTM allows the detection arm to be configured like in standard upright or inverted light microscopy, while removing lateral sample size constraints and extending the accessible imaging depth within a specimen. A simultaneous two-axis scan mode was implemented, enabling synchronous translation of the light sheet and detection focal plane, and facilitating uniform illumination across large tissue volumes.

 

Results: LSTM enables high-resolution imaging over large fields of view with minimal photo-bleaching and phototoxicity. The system produces near-isotropic resolution and demonstrated superior imaging performance in large cleared mouse brains, human brain slices and densely labeled Drosophila larvae compared to standard LSFM approaches. LSTM is particularly effective at minimizing artifacts due to tissue curvature and scattering.

 

Conclusions: LSTM offers a powerful solution to the scalability and resolution challenges of conventional LSFM. Its non-orthogonal geometry enables deep, uniform, high-resolution imaging of large intact tissues, making it especially well-suited for neuroscience and developmental biology applications.