Light Sheet Microscopy-Assisted 3D Analysis of SARS-CoV-2 Infection in the Respiratory Tract of the Ferret Model

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host–pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (>150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and suggest clustering of infection foci in close proximity. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

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3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract

10.1101/2020.10.17.339051 ◽

2020 ◽

Cited By ~ 1

Author(s):

Luca M. Zaeck ◽

David Scheibner ◽

Julia Sehl ◽

Martin Müller ◽

Donata Hoffmann ◽

...

Keyword(s):

Respiratory Tract ◽

3D Visualization ◽

Respiratory Pathogens ◽

Upper Respiratory Tract ◽

Thin Sections ◽

Light Sheet Microscopy ◽

Infection Pattern ◽

Focal Infection ◽

Upper Respiratory

AbstractThe visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host-pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (> 150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and emphasize a distinct focal infection pattern in nasal turbinates. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

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3D visualization of macromolecule synthesis

eLife ◽

10.7554/elife.60354 ◽

2020 ◽

Vol 9 ◽

Author(s):

Timothy J Duerr ◽

Ester Comellas ◽

Eun Kyung Jeon ◽

Johanna E Farkas ◽

Marylou Joetzjer ◽

...

Keyword(s):

3D Visualization ◽

Fluorescent Microscopy ◽

Three Dimensional ◽

Light Sheet ◽

Macromolecular Synthesis ◽

Volumetric Imaging ◽

Processing Pipeline ◽

Powerful Means

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

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High-Resolution 3D Imaging of Virus Infections in Solvent-Cleared Organs: Novel Insights into Virus Replication and Tropism In Vivo

Proceedings ◽

10.3390/proceedings2020050075 ◽

2020 ◽

Vol 50 (1) ◽

pp. 75

Author(s):

Luca Zaeck ◽

Madlin Potratz ◽

Antonia Klein ◽

Conrad M. Freuling ◽

Thomas Müller ◽

...

Keyword(s):

Rabies Virus ◽

Glial Cells ◽

Virus Replication ◽

3D Visualization ◽

Virus Protein ◽

3D Analysis ◽

Virus Infections ◽

Thin Sections ◽

Cellular Environment

The visualization of infection processes in relevant tissues and organs using microscopy methods reveals a unique link between the distribution, tropism, and abundance of pathogens and the physiological structure of the respective organ. To dissect virus replication and the host reaction in vivo at both a global and a single-cell level, conventional 2D imaging approaches can only provide limited insight. However, pathological studies of infected organ material are still mostly restricted to the immunostaining of thin sections from paraffin-embedded or frozen samples. While the 3D analysis of large tissue volumes is possible via laborious serial sectioning, a variety of problems and artifacts remain. Modern immunostaining-compatible tissue clearing techniques allow for the seamless 3D visualization of infection sites in optically cleared thick tissues sections or even entire organs. Benefiting from pure optical slicing, this approach enables the acquisition of multicolor high-volume 3D image stacks for coherent qualitative and quantitative analyses of the infection and its surrounding cellular environment. Here, we demonstrate the utility and power of this methodology by visualizing virus infections in different target tissues. For instance, we reconstructed the cellular context of rabies virus infection sites in mouse brain tissue, allowing a thorough investigation and quantitative analysis of rabies virus cell tropism. The systematic comparison of different rabies viruses with varying pathogenicity revealed a remarkable difference for highly virulent street rabies viruses and attenuated lab strains. While the virus protein expression was readily detectable at a comparable level in both neurons and non-neuronal glial cells from brains of mice infected with street rabies viruses, it was virtually absent in glial cells of lab strain-infected mice. These data provide novel and detailed insights into the pathogenesis of virus infections and substantially contribute to an improved understanding of virus–host interactions in vivo.

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3D Visualization of Macromolecule Synthesis

10.1101/2020.06.24.169300 ◽

2020 ◽

Author(s):

Timothy J. Duerr ◽

Ester Comellas ◽

Eun Kyung Jeon ◽

Johanna E. Farkas ◽

Marylou Joetzjer ◽

...

Keyword(s):

3D Visualization ◽

Fluorescent Microscopy ◽

Three Dimensional ◽

Light Sheet ◽

Macromolecular Synthesis ◽

Volumetric Imaging ◽

Processing Pipeline ◽

Powerful Means

AbstractMeasuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here, we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

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Cell-accurate optical mapping across the entire developing heart

eLife ◽

10.7554/elife.28307 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 24

Author(s):

Michael Weber ◽

Nico Scherf ◽

Alexander M Meyer ◽

Daniela Panáková ◽

Peter Kohl ◽

...

Keyword(s):

High Speed ◽

Cell Function ◽

Three Dimensional ◽

Light Sheet ◽

Tissue Level ◽

In Vivo Studies ◽

Light Sheet Microscopy ◽

Developing Heart ◽

And Function

Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca2+-mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.

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Light sheet fluorescence microscopy as a new method for unbiased three-dimensional analysis of vascular injury

Cardiovascular Research ◽

10.1093/cvr/cvaa037 ◽

2020 ◽

Cited By ~ 4

Author(s):

Nicholas E Buglak ◽

Jennifer Lucitti ◽

Pablo Ariel ◽

Sophie Maiocchi ◽

Francis J Miller ◽

...

Keyword(s):

Carotid Artery ◽

Fluorescence Microscopy ◽

Vascular Injury ◽

Histological Analysis ◽

3D Visualization ◽

Three Dimensional ◽

Light Sheet ◽

3D Analysis ◽

Preclinical Models ◽

Light Sheet Fluorescence Microscopy

Abstract Aims Assessment of preclinical models of vascular disease is paramount in the successful translation of novel treatments. The results of these models have traditionally relied on two-dimensional (2D) histological methodologies. Light sheet fluorescence microscopy (LSFM) is an imaging platform that allows for three-dimensional (3D) visualization of whole organs and tissues. In this study, we describe an improved methodological approach utilizing LSFM for imaging of preclinical vascular injury models while minimizing analysis bias. Methods and results The rat carotid artery segmental pressure-controlled balloon injury and mouse carotid artery ligation injury were performed. Arteries were harvested and processed for LSFM imaging and 3D analysis, as well as for 2D area histological analysis. Artery processing for LSFM imaging did not induce vessel shrinkage or expansion and was reversible by rehydrating the artery, allowing for subsequent sectioning and histological staining a posteriori. By generating a volumetric visualization along the length of the arteries, LSFM imaging provided different analysis modalities including volumetric, area, and radial parameters. Thus, LSFM-imaged arteries provided more precise measurements compared to classic histological analysis. Furthermore, LSFM provided additional information as compared to 2D analysis in demonstrating remodelling of the arterial media in regions of hyperplasia and periadventitial neovascularization around the ligated mouse artery. Conclusion LSFM provides a novel and robust 3D imaging platform for visualizing and quantifying arterial injury in preclinical models. When compared with classic histology, LSFM outperformed traditional methods in precision and quantitative capabilities. LSFM allows for more comprehensive quantitation as compared to traditional histological methodologies, while minimizing user bias associated with area analysis of alternating, 2D histological artery cross-sections.

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Cell-accurate optical mapping across the entire developing heart

10.1101/143057 ◽

2017 ◽

Author(s):

Michael Weber ◽

Nico Scherf ◽

Peter Kohl ◽

Jan Huisken

Keyword(s):

High Speed ◽

Cell Function ◽

Three Dimensional ◽

Light Sheet ◽

Tissue Level ◽

Light Sheet Microscopy ◽

Functional Maturation ◽

Developing Heart ◽

And Function

AbstractOrganogenesis depends on orchestrated interactions between individual cells and morphogenically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighbouring cells, which is established and fine-tuned during development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca2+-mapping of all cells in the entire heart during the looping stage in live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.

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Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

Scientific Reports ◽

10.1038/s41598-020-78640-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Robert J. Francis ◽

Gillian Robb ◽

Lee McCann ◽

Bhagwati Khatri ◽

James Keeble ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Three Dimensional ◽

Large Field ◽

Staining Procedure ◽

3D Analysis ◽

Mycobacterium Tuberculosis Infection ◽

In Vivo Models ◽

Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

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Three-dimensional quantification of twisting in the Arabidopsis petiole

Journal of Plant Research ◽

10.1007/s10265-021-01291-7 ◽

2021 ◽

Author(s):

Yuta Otsuka ◽

Hirokazu Tsukaya

Keyword(s):

Cross Sections ◽

Three Dimensional ◽

Light Sheet ◽

Underlying Mechanism ◽

Two Dimensions ◽

Observation Angle ◽

Differential Growth ◽

Light Sheet Microscopy ◽

Definition Of ◽

Twisting Angle

AbstractOrganisms have a variety of three-dimensional (3D) structures that change over time. These changes include twisting, which is 3D deformation that cannot happen in two dimensions. Twisting is linked to important adaptive functions of organs, such as adjusting the orientation of leaves and flowers in plants to align with environmental stimuli (e.g. light, gravity). Despite its importance, the underlying mechanism for twisting remains to be determined, partly because there is no rigorous method for quantifying the twisting of plant organs. Conventional studies have relied on approximate measurements of the twisting angle in 2D, with arbitrary choices of observation angle. Here, we present the first rigorous quantification of the 3D twisting angles of Arabidopsis petioles based on light sheet microscopy. Mathematical separation of bending and twisting with strict definition of petiole cross-sections were implemented; differences in the spatial distribution of bending and twisting were detected via the quantification of angles along the petiole. Based on the measured values, we discuss that minute degrees of differential growth can result in pronounced twisting in petioles.

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