High Resolution Helium Ion Microscopy of 3T3-L1 Adipocytes - Abstract
Structural changes at the plasma membrane of adipocytes are critical to proper insulin stimulated glucose uptake and thus glucose homeostasis. Insulin stimulates the fusion of insulin responsive glucose transporters (GLUT4) containing vesicles at the plasma membrane in adipocytes. Previous studies have focused on the molecular and cellular events required for GLUT4 vesicle fusion, however the structural changes occurring at the plasma membrane are less well characterized. In order to investigate the changes that occur at the plasma membrane upon insulin stimulation we first had to visualize the plasma membrane surface at the nanometer scale. Helium ion microscopy (HIM) is a new technology that allows for high resolution visualization of nanostructures without the need for sample coating. In order to visualize adipocytes plasma membrane surfaces, we first had to develop a protocol for sample preparation to maintain the detail and contrast of nanostructures for imaging by HIM. Our findings show that our sample preparation protocol allows for the visualization of plasma membrane textures and nanostructures. We further show nano structural details of the inner surface of adipocyte plasma membranes by imaging plasma membrane sheets. Finally we examined 3T3-L1 preadipocytes at various stages of differentiation to visualize the structural changes occurring in the conversion of fibroblastic preadipocytes into mature adipocytes. Our results show the unique membrane textures and nanostructures present on various cell types. Taken together, we established a protocol for sample preparation of preadipocytes and adipocyte for the HIM that allows for high resolution visualization of nanostructures on both the cytoplasmic and exofacial surfaces of the plasma membrane.