LSFM-image z-stack of an optically cleared porcine adipose tissue sample

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.