Nanoscale imaging of untreated mammalian cells in a medium with low radiation damage using scanning electron-assisted dielectric microscopy.

Tomoko Okada,Toshihiko Ogura

doi:10.1038/srep29169

Tomoko Okada, Toshihiko Ogura

Open Access

https://doi.org/10.1038/srep29169

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Abstract

Imaging of untreated living cells in a medium at a nanometre-scale resolution under physiological conditions is a significant challenge. Scanning electron microscopy (SEM) is widely used to observe cells in various atmospheric holders or special equipment. However, untreated biological specimens in aqueous solution generally incur heavy radiation damage from the direct electron beam (EB); and these images exhibit very poor contrast. Therefore, a new method for generating high-contrast images of living cells under physiological conditions without radiation damage has been strongly desired. Here, we demonstrate the first nanoscale observation of living cultured mammalian cells using our newly developed scanning-electron assisted dielectric microscopy (SE-ADM) method with a culture dish holder. Using the difference in relative permittivity between water and specimens, our SE-ADM system aids in the visualisation of untreated biological samples in aqueous solution. In addition, specimens incurred only a low level of radiation damage because the tungsten (W)-coated silicon nitride (SiN) film absorbs irradiated electrons. Untreated cells and organelles are clearly visible in high-contrast and high-resolution images without staining and fixation. Furthermore, our method enables the detection of changes in organelle structures within cells via time-lapse imaging with minimal radiation damage.

Highlights

In this process, biological samples are enclosed in a liquid holder composed of W-coated SiN film and are not directly exposed to EB, minimizing electron radiation damage[17]
After a precise analysis of the first and last images, we found that the same cells under the same conditions exhibited slight changes (Fig. 4a–d, red and blue arrows)
EB used in standard methods causes significant radiation damage to biological samples[2,28,29]

Summary

Introduction

Biological samples are enclosed in a liquid holder composed of W-coated SiN film and are not directly exposed to EB, minimizing electron radiation damage[17]. When the electron beam is used to irradiate the 15-nm W layer, the electrons are scattered and absorbed in this layer; a negative potential arises in the irradiated position. This negative potential is detected from the bottom measurement terminal through the specimen in water[17]. Our method enables the detection of changes in organelle structures within cells via time-lapse imaging with minimal radiation damage

Methods

Results

Conclusion