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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

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.


SEM images of the cell surface of mouse fibroblasts infected with cytomegalovirus. Cells were treated with SSE and imaged directly in SEM. (a) Infected cell after 3 min. (b) Uninfected cell. (c–e) Infected cells after 5, 10 and 30 min, respectively. Scale bars, 2 µm (a), 500 nm (b–e).
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RSOS160887F5: SEM images of the cell surface of mouse fibroblasts infected with cytomegalovirus. Cells were treated with SSE and imaged directly in SEM. (a) Infected cell after 3 min. (b) Uninfected cell. (c–e) Infected cells after 5, 10 and 30 min, respectively. Scale bars, 2 µm (a), 500 nm (b–e).

Mentions: To further investigate the ability of the SSE NanoSuit to improve high-resolution imaging of living cells, we made direct observations of an animal virus infecting cells. Mouse fibroblasts infected with cytomegalovirus were treated with the SSE solution and introduced into the SEM. Images taken 3–5 min after infection showed large numbers of virus particles attached to the cell surface (figure 5a,c). Uninfected cell had no such particles on the cell surface (figure 5b). Interestingly, we observed that the appearance of the cell surface in the SEM changed with time. After 3–5 min virus particles were clearly visible on the cell surface but after 10 and 30 min the virus particles appeared to be taken up by endocytosis while being observed in the SEM (N.B. the temperature in the SEM chamber is 20°C) (figure 5d,e). A similar but more rapid change in cell surface morphology was observed in infected cells, which were fixed at various times after infection and then treated with SSE and observed in the SEM (electronic supplementary material, figure S2b–d). Although the specimens in electronic supplementary material, figure S2b–d were fixed with 4% glutaraldehyde, they remained ‘wet’ even in high vacuum of the SEM owing to the presence of the SSE NanoSuit.Figure 5.


A modified ‘ NanoSuit ® ’ preserves wet samples in high vacuum: direct observations on cells and tissues in field-emission scanning electron microscopy
SEM images of the cell surface of mouse fibroblasts infected with cytomegalovirus. Cells were treated with SSE and imaged directly in SEM. (a) Infected cell after 3 min. (b) Uninfected cell. (c–e) Infected cells after 5, 10 and 30 min, respectively. Scale bars, 2 µm (a), 500 nm (b–e).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOS160887F5: SEM images of the cell surface of mouse fibroblasts infected with cytomegalovirus. Cells were treated with SSE and imaged directly in SEM. (a) Infected cell after 3 min. (b) Uninfected cell. (c–e) Infected cells after 5, 10 and 30 min, respectively. Scale bars, 2 µm (a), 500 nm (b–e).
Mentions: To further investigate the ability of the SSE NanoSuit to improve high-resolution imaging of living cells, we made direct observations of an animal virus infecting cells. Mouse fibroblasts infected with cytomegalovirus were treated with the SSE solution and introduced into the SEM. Images taken 3–5 min after infection showed large numbers of virus particles attached to the cell surface (figure 5a,c). Uninfected cell had no such particles on the cell surface (figure 5b). Interestingly, we observed that the appearance of the cell surface in the SEM changed with time. After 3–5 min virus particles were clearly visible on the cell surface but after 10 and 30 min the virus particles appeared to be taken up by endocytosis while being observed in the SEM (N.B. the temperature in the SEM chamber is 20°C) (figure 5d,e). A similar but more rapid change in cell surface morphology was observed in infected cells, which were fixed at various times after infection and then treated with SSE and observed in the SEM (electronic supplementary material, figure S2b–d). Although the specimens in electronic supplementary material, figure S2b–d were fixed with 4% glutaraldehyde, they remained ‘wet’ even in high vacuum of the SEM owing to the presence of the SSE NanoSuit.Figure 5.

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.