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Imaging lung regeneration by light sheet microscopy.

ABSTRACT: Optical clearing combined with deep imaging of large biological specimen allows organ-wide visualization of cells in three dimensions (3D) to explore regenerative processes in a spatial context. Here, we investigate the dynamics of airway regeneration following toxin-mediated epithelial injury in cleared whole lung preparations by light sheet microscopy. We use a recently developed knock-in mouse strain labeling bronchiolar Club cells (Scgb1a1-mCherry) to define an optimal clearing procedure that efficiently preserves genetically encoded fluorophores. Dehydration in pH-adjusted tert-butanol followed by clearing in ethyl cinnamate maintained maximum mCherry fluorescence while preventing unfavorable background fluorescence. We apply this technique to depict the course of bronchiolar epithelial renewal from an acute injury phase to early and late recovery stages. 3D reconstructions of whole lungs demonstrate near-complete loss of secretory Club cells throughout the entire respiratory tract 3 days post naphthalene (dpn). Multiple foci of regenerating Club cells emerge at 7 dpn, predominantly at airway bifurcations and in distal terminal bronchioles-anatomical regions assumed to harbor distinct stem/progenitor cells subsets. At 21 dpn, clusters of newly formed Club cells have largely expanded, although the bronchiolar epithelial lining continues to regenerate. This study identifies regional stem cell niches as starting points for epithelial recovery, underscores the enormous regenerative capacity of the respiratory epithelium and demonstrates the power of whole lung 3D imaging for evaluating the extent of pulmonary damage and subsequent repair processes.

SUBMITTER: Salwig I

PROVIDER: S-EPMC7910255 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Imaging lung regeneration by light sheet microscopy.

Salwig Isabelle I Spitznagel Birgit B Wiesnet Marion M Braun Thomas T

Histochemistry and cell biology 20200719 2

Optical clearing combined with deep imaging of large biological specimen allows organ-wide visualization of cells in three dimensions (3D) to explore regenerative processes in a spatial context. Here, we investigate the dynamics of airway regeneration following toxin-mediated epithelial injury in cleared whole lung preparations by light sheet microscopy. We use a recently developed knock-in mouse strain labeling bronchiolar Club cells (Scgb1a1-mCherry) to define an optimal clearing procedure tha ...[more]

PMID: 32685990

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Project description:Live observation of biological phenomena in the context of living organisms can provide important insights in the mechanisms of these phenomena. However, the spatially complex and dynamic physiology of multicellular organisms can be a challenging environment to make observations with fluorescence microscopy. Due to the illumination of out-of-focus planes, confocal and particularly widefield fluorescence microscopy suffer from low signal-to-background ratio (SBR), photo toxicity and bleaching of fluorescent probes. In light-sheet microscopy (LSM), solely the focal plane of the detection objective is illuminated, minimising out-of-focus fluorescence and photobleaching, thereby enhancing SBR, allowing for low laser intensities and longer acquisition periods. Here we present a straightforward light-sheet microscope with a 1.0-NA detection objective and a fast sample-positioning stage that allows for four degrees of freedom. By imaging the sensory cilia and nervous system of living young adult C. elegans, we demonstrate that the instrument is well suited for relatively fast, volumetric imaging of larger (hundreds of micrometres cubed) living samples. These experiments demonstrate that such an instrument provides a valuable addition to commonly used widefield and confocal fluorescence microscopes. LAY DESCRIPTION: In fluorescence microscopy, sharp images can only be obtained when the light obtained from the section of the image that is in focus is not overwhelmed by light emerging from elsewhere. In this paper, we present a light-sheet fluorescence microscope, based on the OpenSPIM initiative, with a magnification of 90× and a sensitive sample positioning stage that allows for fast controlled linear movement and rotation. In a light-sheet microscope (LSM), the sample is illuminated from the side, compared to the direction of detection, limiting illumination only to the part of the sample that is imaged in the focal plane (general resources: Wikipedia or MicroscopyU). This does not only limit background noise, but also reduces damage to the sample due to phototoxicity. This makes a LSM particularly suitable for imaging living samples at high resolution, in three dimensions, over long periods of time. Our instrument was specifically designed for imaging adult C. elegans nematodes. We show here how the instrument compares to a standard epifluorescence microscope, imaging neuronal structures in the animals. The instrument proved well suited for fast volumetric imaging of larger cellular structures such as C. elegans neuronal cell bodies. Our experiments show that the instrument provides a valuable addition to widefield and confocal fluorescence microscopes commonly used to image adult C. elegans.

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Imaging lung regeneration by light sheet microscopy.

Publications

Imaging lung regeneration by light sheet microscopy.

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