scholarly journals Selective Plane Illumination Microscopy and Computing Reveal Differential Obliteration of Retinal Vascular Plexuses

2020 ◽  
Author(s):  
Chih-Chiang Chang ◽  
Alison Chu ◽  
Scott Meyer ◽  
Michel M. Sun ◽  
Parinaz Abiri ◽  
...  
Keyword(s):  
Principal Component ◽  
Light Sheet ◽  
Murine Models ◽  
Euler Numbers ◽  
Oxygen Induced Retinopathy ◽  
Topological Network ◽  
Vascular Plexuses ◽  
Computational Analyses ◽  
Light Sheet Fluorescence Microscopy ◽  
Vascular Branching

ABSTRACTMurine models of visual impairment provide micro-vascular insights into the 3-D network disarray in retinopathy. Current imaging and analysis tend to be confined to the 2-D retinal vasculature. We hereby integrated selective plane illumination imaging or known as light-sheet fluorescence microscopy (LSFM) with dual-illumination, followed by computational analyses, to reveal the topological network of vertical sprouts bridging the primary and secondary plexuses in a postnatal mouse model of oxygen-induced retinopathy (OIR). We revealed a preferential obliteration of the secondary plexus and bridging vessels despite a relatively unscathed primary plexus. We compared the local versus global vascular connectivity using clustering coefficients and Euler numbers, respectively. The global vascular connectivity in hyperoxia-exposed retinas was significantly reduced (p < 0.05, n = 5 vs. normoxia), whereas the local connectivity was preserved (p > 0.05, n = 5 vs. normoxia). We further applied principal component analysis (PCA) to automatically segment the vertical sprouts, corroborating the preferential obliteration of the interconnection between vertical sprouts and secondary plexuses that were accompanied with impaired vascular branching and connectivity, and reduced vessel volumes and lengths (p < 0.05, n=5 vs. normoxia). Thus, integration of 3-D selective plane illumination with computational analyses allows for early detection of global and spatially-specific vaso-obliteration, but preserved local reticular structure in response to hyperoxia-induced retinopathy.

scholarly journals 3D light sheet fluorescence microscopy of lungs to dissect local host immune -Aspergillus fumigatusinteractions

2019 ◽  
Author(s):  
Jorge Amich ◽  
Zeinab Mokhtari ◽  
Marlene Strobel ◽  
Elena Vialetto ◽  
Natarajaswamy Kalleda ◽  
...  
Keyword(s):  
Animal Models ◽  
Fluorescence Microscopy ◽  
Immune Cell ◽  
Invasive Pulmonary Aspergillosis ◽  
Fungal Growth ◽  
Light Sheet ◽  
Murine Models ◽  
Pulmonary Aspergillosis ◽  
Local Host ◽  
Light Sheet Fluorescence Microscopy

ABSTRACTAspergillus fumigatusis an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment and progression are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are paramount to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in the desired intra-alveolar deposition, as more than 60% of fungal growth occurred outside of the alveolar space. Hence, LSFM allows for more rigorous characterization of murine models of invasive pulmonary aspergillosis and pinpointing their strengths and limitations.IMPORTANCEThe use of animal models of infection is essential to advance our understanding of complex host-pathogen interactions that take place duringAspergillus fumigatuslung infections. As in the case of humans, mice need to be immunosuppressed to become susceptible to invasive pulmonary aspergillosis, the most serious infection caused byA. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis (IPA). However, the precise consequences that each immunosuppressive model has on the local immune populations and for fungal growth are not completely understood. Here we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution, to pin down the scenario commonly used IPA models. Our results will be valuable to optimize and refine animal models to maximize their use in future research.VISUAL ABSTRACTQuantitative light sheet fluorescence microscopy to dissect local host-pathogen interactions in the lung afterA. fumigatusairway infection.

scholarly journals Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jorge Amich ◽  
Zeinab Mokhtari ◽  
Marlene Strobel ◽  
Elena Vialetto ◽  
Dalia Sheta ◽  
...  
Keyword(s):  
Animal Models ◽  
Fluorescence Microscopy ◽  
Aspergillus Fumigatus ◽  
Invasive Pulmonary Aspergillosis ◽  
Fungal Growth ◽  
Light Sheet ◽  
Murine Models ◽  
Pulmonary Aspergillosis ◽  
Content Type ◽  
Light Sheet Fluorescence Microscopy

ABSTRACT Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations. IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus. There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

scholarly journals Cytotoxic effects and tolerability of gemcitabine and axitinib in a xenograft model for c-myc amplified medulloblastoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Stefanie Schwinn ◽  
Zeinab Mokhtari ◽  
Sina Thusek ◽  
Theresa Schneider ◽  
Anna-Leena Sirén ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Tumor Growth ◽  
Intensive Therapy ◽  
Xenograft Model ◽  
Light Sheet ◽  
Tumor Control ◽  
Orthotopic Model ◽  
Group 3 ◽  
Light Sheet Fluorescence Microscopy

AbstractMedulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.

Investigating safe excitation in Light Sheet Fluorescence Microscopy

2021 ◽  
Vol 84 ◽  
pp. 296
Author(s):  
Gideon Oluniran ◽  
James Blackwell ◽  
Emmanuel Reynaud ◽  
Marcin Krasny ◽  
Niall Colgan ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Light Sheet ◽  
Light Sheet Fluorescence Microscopy

scholarly journals Light sheet fluorescence microscopy of optically cleared brains for studying the glymphatic system

2020 ◽  
Vol 40 (10) ◽  
pp. 1975-1986
Author(s):  
Nicholas B Bèchet ◽  
Tekla M Kylkilahti ◽  
Bengt Mattsson ◽  
Martina Petrasova ◽  
Nagesh C Shanbhag ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluid Transport ◽  
Light Sheet ◽  
Brain Parenchyma ◽  
Sleep State ◽  
Glymphatic System ◽  
Light Sheet Microscopy ◽  
Highly Active ◽  
Quantitative Analyses ◽  
Light Sheet Fluorescence Microscopy

Fluid transport in the perivascular space by the glia-lymphatic (glymphatic) system is important for the removal of solutes from the brain parenchyma, including peptides such as amyloid-beta which are implicated in the pathogenesis of Alzheimer’s disease. The glymphatic system is highly active in the sleep state and under the influence of certain of anaesthetics, while it is suppressed in the awake state and by other anaesthetics. Here we investigated whether light sheet fluorescence microscopy of whole optically cleared murine brains was capable of detecting glymphatic differences in sleep- and awake-mimicking anaesthesia, respectively. Using light-sheet imaging of whole brains, we found anaesthetic-dependent cerebrospinal fluid (CSF) influx differences, including reduced tracer influx along tertiary branches of the middle cerebral artery and reduced influx along dorsal and anterior penetrating arterioles, in the awake-mimicking anaesthesia. This study establishes that light sheet microscopy of optically cleared brains is feasible for quantitative analyses and can provide images of the entire glymphatic system in whole brains.

scholarly journals Enhancement and Segmentation Workflow for the Developing Zebrafish Vasculature

2019 ◽  
Vol 5 (1) ◽  
pp. 14 ◽  
Author(s):  
Elisabeth Kugler ◽  
Karen Plant ◽  
Timothy Chico ◽  
Paul Armitage
Keyword(s):  
Open Source Software ◽  
Light Sheet ◽  
Visual Assessment ◽  
Cardiovascular Development ◽  
Fluorescent Reporter ◽  
Enhancement Method ◽  
Vertebrate Model ◽  
Vascular Enhancement ◽  
Light Sheet Fluorescence Microscopy

Zebrafish have become an established in vivo vertebrate model to study cardiovascular development and disease. However, most published studies of the zebrafish vascular architecture rely on subjective visual assessment, rather than objective quantification. In this paper, we used state-of-the-art light sheet fluorescence microscopy to visualize the vasculature in transgenic fluorescent reporter zebrafish. Analysis of image quality, vascular enhancement methods, and segmentation approaches were performed in the framework of the open-source software Fiji to allow dissemination and reproducibility. Here, we build on a previously developed image processing pipeline; evaluate its applicability to a wider range of data; apply and evaluate an alternative vascular enhancement method; and, finally, suggest a work-flow for successful segmentation of the embryonic zebrafish vasculature.

FEATURES

2016 ◽  
Vol 20 (12) ◽  
pp. 12-22
Keyword(s):  
Medical Imaging ◽  
Fluorescence Microscopy ◽  
Fluorescence Spectroscopy ◽  
Medical Images ◽  
Light Sheet ◽  
The Future ◽  
Light Sheet Fluorescence Microscopy

Scanning the Future of Medical Imaging Putting Numbers into Biology: The Combination of Light Sheet Fluorescence Microscopy and Fluorescence Spectroscopy Abyss Processing – Exploring the Deep in Medical Images

scholarly journals Spatial and Temporal Features of the Growth of a Bacterial Species Colonizing the Zebrafish Gut

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Matthew Jemielita ◽  
Michael J. Taormina ◽  
Adam R. Burns ◽  
Jennifer S. Hampton ◽  
Annah S. Rolig ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Structure ◽  
Growth Kinetics ◽  
Spatial Organization ◽  
Temporal Dynamics ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Light Sheet ◽  
Logistic Growth ◽  
Light Sheet Fluorescence Microscopy

ABSTRACTThe vertebrate intestine is home to microbial ecosystems that play key roles in host development and health. Little is known about the spatial and temporal dynamics of these microbial communities, limiting our understanding of fundamental properties, such as their mechanisms of growth, propagation, and persistence. To address this, we inoculated initially germ-free zebrafish larvae with fluorescently labeled strains of anAeromonasspecies, representing an abundant genus in the zebrafish gut. Using light sheet fluorescence microscopy to obtain three-dimensional images spanning the gut, we quantified the entire bacterial load, as founding populations grew from tens to tens of thousands of cells over several hours. The data yield the first ever measurements of the growth kinetics of a microbial species inside a live vertebrate intestine and show dynamics that robustly fit a logistic growth model. Intriguingly, bacteria were nonuniformly distributed throughout the gut, and bacterial aggregates showed considerably higher growth rates than did discrete individuals. The form of aggregate growth indicates intrinsically higher division rates for clustered bacteria, rather than surface-mediated agglomeration onto clusters. Thus, the spatial organization of gut bacteria both relative to the host and to each other impacts overall growth kinetics, suggesting that spatial characterizations will be an important input to predictive models of host-associated microbial community assembly.IMPORTANCEOur intestines are home to vast numbers of microbes that influence many aspects of health and disease. Though we now know a great deal about the constituents of the gut microbiota, we understand very little about their spatial structure and temporal dynamics in humans or in any animal: how microbial populations establish themselves, grow, fluctuate, and persist. To address this, we made use of a model organism, the zebrafish, and a new optical imaging technique, light sheet fluorescence microscopy, to visualize for the first time the colonization of a live, vertebrate gut by specific bacteria with sufficient resolution to quantify the population over a range from a few individuals to tens of thousands of bacterial cells. Our results provide unprecedented measures of bacterial growth kinetics and also show the influence of spatial structure on bacterial populations, which can be revealed only by direct imaging.

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