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

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ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Comparison of images of human fibroblast treated with the SSE solution (a–d) and prepared by traditional methods (e–h). Fibres of the cells (b,f), nucleus (c,g), high magnifications of the cell surface (d,h). In traditional methods, some cells show protrusions on the surface (arrow in h). Scale bars, 20 µm (a,e), 10 µm (b,c,f,g), 5 µm (d,h).
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RSOS160887F2: Comparison of images of human fibroblast treated with the SSE solution (a–d) and prepared by traditional methods (e–h). Fibres of the cells (b,f), nucleus (c,g), high magnifications of the cell surface (d,h). In traditional methods, some cells show protrusions on the surface (arrow in h). Scale bars, 20 µm (a,e), 10 µm (b,c,f,g), 5 µm (d,h).

Mentions: In addition to the apparent barrier effect, we found that the fine structure of the specimens treated with SSE solution was completely different from that of conventionally prepared, i.e. fixed and metal coated, specimens. Following conventional preparation, shrinkage of tissues and cells was inevitable owing to dehydration (figures 1d,h, 2e–h). By comparison, the overall morphology of SSE-treated specimens seemed well preserved and intact (figures 1c,g, 2a–d).Figure 2.


A modified ‘ NanoSuit ® ’ preserves wet samples in high vacuum: direct observations on cells and tissues in field-emission scanning electron microscopy
Comparison of images of human fibroblast treated with the SSE solution (a–d) and prepared by traditional methods (e–h). Fibres of the cells (b,f), nucleus (c,g), high magnifications of the cell surface (d,h). In traditional methods, some cells show protrusions on the surface (arrow in h). Scale bars, 20 µm (a,e), 10 µm (b,c,f,g), 5 µm (d,h).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383832&req=5

RSOS160887F2: Comparison of images of human fibroblast treated with the SSE solution (a–d) and prepared by traditional methods (e–h). Fibres of the cells (b,f), nucleus (c,g), high magnifications of the cell surface (d,h). In traditional methods, some cells show protrusions on the surface (arrow in h). Scale bars, 20 µm (a,e), 10 µm (b,c,f,g), 5 µm (d,h).
Mentions: In addition to the apparent barrier effect, we found that the fine structure of the specimens treated with SSE solution was completely different from that of conventionally prepared, i.e. fixed and metal coated, specimens. Following conventional preparation, shrinkage of tissues and cells was inevitable owing to dehydration (figures 1d,h, 2e–h). By comparison, the overall morphology of SSE-treated specimens seemed well preserved and intact (figures 1c,g, 2a–d).Figure 2.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus