DOI: 10.5061/dryad.8gtht76nt
title: LSFM-image z-stack of an optically cleared porcine adipose tissue sample

description: In translational obesity research, objective assessment of adipocyte sizes
 and numbers is essential to characterize histomorphological alterations
 linked to obesity, and to evaluate the efficacies of experimental
 medicinal or dietetic interventions. Design-based quantitative
 stereological techniques based on the analysis of 2D-histological sections
 provide unbiased estimates of relevant 3D-parameters of adipocyte
 morphology, but often involve complex and time-consuming tissue processing
 and analysis steps. Here we report the application of direct 3D light
 sheet fluorescence microscopy (LSFM) for effective and accurate analysis
 of adipocyte volumes and numbers in optically cleared adipose tissue
 samples from a porcine model of diet-induced obesity (DIO). Subcutaneous
 and visceral adipose tissue samples from DIO-minipigs and lean controls
 were systematically randomly sampled, optically cleared with 3DISCO
 (3-dimensional imaging of solvent cleared organs), stained with eosin, and
 subjected to LSFM for detection of adipocyte cell membrane
 autofluorescence. Individual adipocytes were unbiasedly sampled in digital
 3D reconstructions of the adipose tissue samples, and their individual
 cell volumes were directly measured by automated digital image analysis.
 Adipocyte numbers and mean volumes obtained by LSFM analysis did not
 significantly differ from the corresponding values obtained by unbiased
 quantitative stereological analysis techniques performed on the same
 samples, thus proving the applicability of LSFM for efficient analysis of
 relevant morphological adipocyte parameters. The results of the present
 study demonstrate an adipose tissue depot-specific plasticity of adipocyte
 growth responses to nutrient oversupply. This was characterized by an
 exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes
 in subcutaneous fat tissue depots also displayed a marked (hyperplastic)
 increase in cell number. LSFM allows for accurate and efficient
 determination of relevant quantitative morphological adipocyte parameters.
 The applied stereological methods and LSFM protocols are described in
 detail and can serve as a guideline for unbiased quantitative
 morphological analyses of adipocytes in other studies and species. The
 dataset “LSFM-image z-stack of an optically cleared porcine adipose tissue
 sample” (doi:10.5061/dryad.8gtht76nt) is a Light-sheet fluorescence
 microscopy (LSFM) image z-stack consisting of 181 image files that were
 acquired in an 3DISCO-cleared, eosin-stained porcine subcutaneous adipose
 tissue sample of an obese Göttingen minipig from the study by Theobalt et
 al. (2021). The dataset is thought to serve as a training dataset for
 unbiased sampling and volume analysis of adipocytes in 3D LSFM image
 reconstructions, using the arivis Vision4D imaging and analysis software.
description: The images were acquired at Ex/Em: 520/40nm/585/40nm with a
 z-step height of 5 µm. Further details on the used LSFM hardware con be
 found in the publication. A detailed instruction for how to analyze the
 data is presented in the Supporting Information S1 file.
description: <b>Details that may influence reuse or replication
 efforts</b> Do not change the names of the
 image-files, as this is important for the sequence of the images in the
 image z-stack. Follow the instructions provided in the Supporting
 Information S1 file “<i>Step-by-step protocol for unbiased sampling
 and volume analysis of adipocytes in 3D LSFM image reconstructions, using
 the arivis Vision4D imaging and analysis software</i>”.
 <b>Specialized software</b> arivis
 Vision4D (arivis, Germany) imaging and analysis software
 <i>(</i><i>https://www.arivis.com/en/</i><i>)</i> <b>Description of file(s)</b> The image z-stack consists of 181 images. The images are named in a sequential order starting with the first image being named: “12-13-51_473_17_obese_Ro_3_UltraII_C00_xyz-Table Z0070.ome” and with the last image named :”12-13-51_473_17_obese_Ro_3_UltraII_C00_xyz-Table Z0250.ome”. The sequence of the images is defined by their name. For upload of the images into the arivis Vision4D software, calibration of x-, y-, and z-dimensions (pixel sizes), 3D-image reconstruction, and quantitative morphological analysis of adipocytes please refer to the informations provided in the Supporting Information S1 file “<i>Step-by-step protocol for unbiased sampling and volume analysis of adipocytes in 3D LSFM image reconstructions, using the arivis Vision4D imaging and analysis software</i>”.