Three-Dimensional Visualization of the Lymphatic Vasculature

Dierkes, Cathrin; Scherzinger, Aaron; Kiefer, Friedemann

doi:10.1007/978-1-4939-8712-2_1

Cited by 8 publications

(10 citation statements)

References 13 publications

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“…The results reported here were obtained with a basic single objective configuration, illuminated by a dual sided light sheet and we visualized image stacks using our proprietary volume-rendering framework Voreen ( Meyer-Spradow et al, 2009 ; Dierkes et al, 2018 ). We analyzed the same immunostained liver biopsy specimen with LSFM that was later also imaged using OPT.…”

Section: Resultsmentioning

confidence: 99%

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies

et al. 2021

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The liver as the largest organ in the human body is composed of a complex macroscopic and microscopic architecture that supports its indispensable function to maintain physiological homeostasis. Optical imaging of the human liver is particularly challenging because of the need to cover length scales across 7 orders of magnitude (from the centimeter scale to the nanometer scale) in order to fully assess the ultrastructure of the entire organ down to the subcellular scale and probe its physiological function. This task becomes even more challenging the deeper within the organ one hopes to image, because of the strong absorption and scattering of visible light by the liver. Here, we demonstrate how optical imaging methods utilizing highly specific fluorescent labels, as well as label-free optical methods can seamlessly cover this entire size range in excised, fixed human liver tissue and we exemplify this by reconstructing the biliary tree in three-dimensional space. Imaging of tissue beyond approximately 0.5 mm length requires optical clearing of the human liver. We present the successful use of optical projection tomography and light-sheet fluorescence microscopy to derive information about the liver architecture on the millimeter scale. The intermediate size range is covered using label-free structural and chemically sensitive methods, such as second harmonic generation and coherent anti-Stokes Raman scattering microscopy. Laser-scanning confocal microscopy extends the resolution to the nanoscale, allowing us to ultimately image individual liver sinusoidal endothelial cells and their fenestrations by super-resolution structured illumination microscopy. This allowed us to visualize the human hepatobiliary system in 3D down to the cellular level, which indicates that reticular biliary networks communicate with portal bile ducts via single or a few ductuli. Non-linear optical microscopy enabled us to identify fibrotic regions extending from the portal field to the parenchyma, along with microvesicular steatosis in liver biopsies from an older patient. Lastly, super-resolution microscopy allowed us to visualize and determine the size distribution of fenestrations in human liver sinusoidal endothelial cells for the first time under aqueous conditions. Thus, this proof-of-concept study allows us to demonstrate, how, in combination, these techniques open up a new chapter in liver biopsy analysis.

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Section: Resultsmentioning

confidence: 99%

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies

et al. 2021

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“…Therefore, we used wholemount immunostaining with an RFP antibody to counterstain all tissue specimen derived from a Vegfr3 -tdTomato pup at P5 that were subsequently cleared following the BABB protocol (Fig 6). 3D-volume rendering of image stacks was performed using the open source visualization software package Voreen [37, 38]. In the lung a highly branched lymphatic network was observed lancing both lobes to the level of the terminal bronchioles (Fig 6 A-C).…”

Section: Resultsmentioning

confidence: 99%

“…For image acquisition, Z-steps 3 μm were chosen. 3D reconstruction and analysis of ultramicroscopy stacks were performed by using the volume rendering software Voreen [37, 48].…”

Section: Methodsmentioning

confidence: 99%

Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities

Redder

Kirschnick

Hägerling

et al. 2021

Preprint

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Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements. We introduce here the BAC-transgenic mouse reporter strain Vegfr3 -tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements. Vegfr3 -tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells.

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“…We applied this hierarchical random walker approach first to a 60 GB data set that covered 52 mm 3 of a BABBcleared, anti-PECAM1-stained mouse kidney. Labeling of seeds was done in a similar fashion as described by Prassni et al (Prassni et al, 2010) using a multi-view interface of the data: The input data set and the intermediate segmentation results were rendered in 3D using the OpenCL raycaster of Voreen (Figure 7 and Video S3) (Dierkes et al, 2018). Vessels of a total length of 160 mm enclosing a volume of 0.35 mm 3 and comprising the renal vasculature to the level of the interlobular vasculature were segmented interactively (Figures 7B, 7E, and 7H).…”

Section: Semi-automatic Segmentation and Analysis Of Vascular Network In Large Light Sheet Microscopy-based Data Setsmentioning

confidence: 99%

Rapid methods for the evaluation of fluorescent reporters in tissue clearing and the segmentation of large vascular structures

et al. 2021

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Three-Dimensional Visualization of the Lymphatic Vasculature

Cited by 8 publications

References 13 publications

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies

Multiscale and Multimodal Optical Imaging of the Ultrastructure of Human Liver Biopsies

Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities

Rapid methods for the evaluation of fluorescent reporters in tissue clearing and the segmentation of large vascular structures

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