A modified 'NanoSuit®' preserves wet samples in high vacuum: direct observations on cells and tissues in field-emission scanning electron microscopy.

Although field-emission scanning electron microscopy (FE-SEM) has proven very useful in biomedical research, the high vacuum required (10(-3) to 10(-7) Pa) precludes direct observations of living cells and tissues at high resolution and often produces unwanted structural changes. We have previously described a method that allows the investigator to keep a variety of insect larvae alive in the high vacuum environment of the electron microscope by encasing the organisms in a thin, vacuum-proof suit, the 'NanoSuit®'. However, it was impossible to protect wet tissues freshly excised from intact organisms or cultured cells. Here we describe an improved 'NanoSuit' technique to overcome this limitation. We protected the specimens with a surface shield enhancer (SSE) solution that consists of glycerine and electrolytes and found that the fine structure of the SSE-treated specimens is superior to that of conventionally prepared specimens. The SSE-based NanoSuit affords a much stronger barrier to gas and/or liquid loss than the previous NanoSuit did and, since it allows more detailed images, it could significantly help to elucidate the 'real' organization of cells and their functions.