Large Field/High Resolution Backscatter Scanning Electron Microscopy of the Paraventricular Nucleus of the Hypothalamus

Abstract

The paraventricular nucleus of the hypothalamus (PVN) is critical in autonomic and endocrine control. A significant body of work has indicated that dysregulation in the PVN is associated with a variety of cardiovascular, metabolic, and reproductive diseases. Changes in the anatomical structure of brain networks leads to functional alterations. In this context, electron microscopy (EM) can provide nanometer resolution of normal/abnormal brain structures and individual organelles. However, an inherent limitation of traditional EM is the random sampling of an object within a large region of interest (i.e. PVN). This limited field of view approach is like a fishing expedition in that it does not allow for localization of subcellular changes in relation to the overall organization of the nucleus. To overcome this, here we propose an approach that utilizes large‐field of view high‐resolution scanning electron microscopy (SEM) of the PVN. This technique includes the development of zoomable, Google map‐like image sets to integrate cellular/organelle location within the overall PVN with ultrastructural detail. Perfused and post‐fixed brains from 16‐week‐old male C57Bl/6J mice were coronally sectioned into six 300μm slices containing the rostral to caudal extent of the PVN. The sections were fixed in osmium tetroxide (1%) and potassium ferrocyanide (1%), infiltrated with uranyl acetate (1%), dehydrated, and flat embedded in resin. Ultrathin sections (70 nm) containing the PVN were obtained and trimmed to 2×2mm. Sections were then placed on silicon wafers for imaging in a FEI Helios NanoLab™ 660 focused ion beam‐SEM equipped with a retractable concentric backscattering detector. As a first step (Figure 1A), the entire section was scanned and stitched at a low magnification (1000x). The low‐resolution data was used to precisely navigate to the area of the PVN. Subsequently, a higher resolution image of the PVN with an 82.88μm horizontal field width was acquired (10000x, 3072×2048pixels, 26.97 nm pixel size) for cellular identification and some ultrastructural detail (Figure B). Final high magnification images (80000x) with a horizontal field width of 5.18μm were taken within the PVN for imaging of single cells (Figure C). Using this approach, high‐magnification images were obtained with the same intensity and quality as images achieved with traditional transmission EM. All neuronal structures and subsynaptic features were readily identifiable, and could be placed within the overall anatomical construct of the PVN (Figure ). Collectively, this methodological advancement has the potential to facilitate robust neuroscience discovery into the PVN by relating high‐resolution data (neurons, glia, synapses, organelles etc) to a very precise and specific functional domain and context.Support or Funding Information1R01HL141393A) Representative unilateral PVN and surrounding regions imaged at a relatively low magnification (1000x) with large field of view SEM. B) A higher resolution (10000x) image of a portion of the PVN (red outline) was then obtained by using the image in A to navigate. The blue outline in A indicates the location of this image. C) High‐resolution (80000x) image of a single neuron within the PVN. The pink box in B indicates the anatomical location of this neuron in the context of the overall PVN.Figure 1