ECE2022 Eposter Presentations Diabetes, Obesity, Metabolism and Nutrition (318 abstracts)
Imidazole-osmium reduces elution of lipids from cryofixed rat hepatic tissue for ultrastructural analysis
Sabine Dürr 1 , Siegfried Reipert 2 , Martin Krššák 3 , Cécile Philippe 4 , Viktoria Ehret 3 , Matthias Luft 5,6 , Arno Schintlmeister 7 , Thomas Scherer 3 & Clemens Fürnsinn 3
1Medical University of Vienna, Wien, Austria; 2University of Vienna, Core Facility Cell Imaging and Ultrastructural Research, Wien, Austria; 3Medical University of Vienna, Department of Medicine III, Wien, Austria; 4Medical University of Vienna, Department of Biomedical Imaging and Image-Guided Therapy, Wien, Austria; 5Medical University of Vienna, Department of Plastic, Reconstructive and Aesthetic Surgery, Wien, Austria; 6Karl Landsteiner University of Health Sciences, Department of Plastic, Aesthetic and Reconstructive Surgery, St. Poelten, Austria; 7University of Vienna, Wien, Austria
Introduction: Transmission electron microscopy (TEM) is the main tool for studying the ultrastructural properties of metabolically diseased tissue. For impactful micrographs as well as for interpretation of TEM specimens, the integrity of cellular components is crucial. Elution of substances is a major problem in TEM preparation with no universal solution found so far. Investigating fatty liver disease in a rat model, an important factor is the depiction of undamaged lipid droplets. Imidazole, a highly polar heterocyclic compound, is hypothesized to enhance the binding ability of osmium tetroxide. This study aimed to investigate the effect of imidazole-osmium application before cryopreparation to prevent elution from lipid droplets.
Methods: Perfusion-fixed (4.5% phosphate buffered formaldehyde, pH 7) hepatic tissue from male Sprague-Dawley rats (n = 6; 8 weeks old) on high fat diet (60% of calories as fat; fed for 4 weeks) was processed for TEM. Prior to the procedure of high pressure freezing and freeze substitution, samples (n = 3) were pre-exposed to 1% OsO<ce:inf>4</ce:inf> in 0.1M imidazole for 30 min. For comparison, a control group was not subject to such pre-exposure (n = 3). Specimens were embedded in Agar 100 resin, and the ultrastructure was analyzed in ultrathin sections (70 nm, Leica EM UC7; ZEISS Libra 120 TEM).
Results: Screening a multitude of ultrathin sections from steatotic rat livers from three separate animals revealed expletive electron dense material within the membrane of lipid droplets in imidazole-osmium pre-treated tissue. At variance to this, the conventional approach without imidazole pre-treatment displayed translucent areas with minimal granular content.
Conclusion: The chemical bond between osmium tetroxide and imidazole obviously reduced the elution of contrastable lipid molecules from lipid droplets prior to preparation and high pressure freezing with freeze substitution. This procedure promises a major advantage in the ultrastructural study of fat accumulation in hepatic tissue. Further investigations including NanoSIMS (Nanoscale secondary ion Mass Spectrometry) will be conducted to confirm this hypothesis.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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