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.

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.

Details that may influence reuse or replication efforts

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 “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”.

Specialized software

arivis Vision4D (arivis, Germany) imaging and analysis software (https://www.arivis.com/en/)

Description of file(s)

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 “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”.