Large-Area Fluorescence and Electron Microscopic Correlative Imaging With Multibeam Scanning Electron Microscopy.

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.

Inspection of skin hemodynamics with hyperspectral camera.

Using hyperspectral imaging techniques, which enable us to simultaneously obtain spatial and wavelength information, we have improved upon the newly developed hyperspectral camera to develop a method of observing changes in skin melanin levels and hemodynamics over time. As an evaluation of this method, we measured the skin diffuse reflectance spectrum of the human middle finger in an experiment of blood flow blockage in the brachial region. The changes in skin hemodynamics observed through this method match the behavior expected based on clinical knowledge, and also show an extremely high correlation with results obtained by the Erythema Index, which is used to make similar calculations from a limited number of wavelengths.