Correlative light-electron microscopy in liquid using an inverted SEM (ASEM).

In atmospheric scanning electron microscope (ASEM), the inverted scanning electron microscope (SEM) observes the wet sample from below, while an optical microscope observes it from above simultaneously. The ASEM sample holder has a disposable dish shape with a silicon nitride film window at the bottom. It can be coated variously for the primary-culture of substrate-sensitive cells; primary cells were cultured in a few milliliters of culture medium in a stable incubator environment. For the inverted SEM observation, cells and the excised tissue blocks were aldehyde-fixed, immersed in radical scavenger solution, and observed at minimum electron dose. Neural networking, axonal segmentation, proplatelet-formation and phagocytosis, and Fas expression in embryonic stem cells were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. By exploiting optical microscopy, the region of interest of organ can be found from the wide area, and the cells and organelle were successfully examined at high resolution by the following scanning electron microscopy. We successfully visualized islet of Langerhans, blood microvessels, neuronal endplate, and bacterial flora on stomach epidermal surfaces. Bacterial biofilms and the typical structural features including "leg complex" of mycoplasma were visualized by exploiting CLEM of ASEM. Based on these studies, ASEM correlative microscopy promises to allow the research of various mesoscopic-scale biological phenomena in the near future.