| Summary: | 博士 === 國立清華大學 === 材料科學工程學系 === 98 === A novel and disposable microchip (named as K-kit) with electron-transparent SiO2 nano-membranes was developed using microelectromechanical system techniques and used as a specimen kit for in situ imaging of living organisms in an aqueous condition by transmission electron microscopy (TEM) without equipment modification. The resolving power of living bacterial cells negatively stained with phosphotungstic acid was theoretically calculated to be 2.2 nm in K-kit at incident current density of 30 pA/cm2. Experimentally, this K-kit which can enclose aqueous specimens enabled the successful TEM observation of living Escherichia coli cells, the 8-18 nm type 3 fimbriae of living Klebsiella pneumoniae, and the tellurite reduction process in K. pneumoniae in-situ.
The survival ratio of K. pneumoniae sealed in the K-kit for 12 h exceeded 80% before TEM imaging. Besides, the viability of bacterial cells sealed in the K-kits during TEM electron irradiation was examined. The K. pneumoniae (gram-negative bacteria) and Saccharomyces cerevisiae (yeast cells) can stay alive in K-kit after continuous TEM imaging for up to 14 s and 42 s, respectively. Utilizing the measurement by electron energy-loss spectroscopy, the total electron energy of 329.1 eV nm-3 and 405.3 eV nm-3 dissipated in K. pneumoniae and S. cerevisiae were calculated, respectively. By the comparison of their surface structures, S. cerevisiae cells exhibit more dense-packed and covalently linked cell walls than the loosely attached capsular polysaccharides on the outer membrane of K. pneumoniae. It is possible that K. pneumoniae is more susceptible to electron beam irradiation compared to S. cerevisiae.
Furthermore, the bio-reaction, tellurite reduction in K. pneumonia, was in situ monitored by TEM for 12.5 h through the use of the K-kit. The different tellurite reduction profiles in cells grown in aerobic and anaerobic environments can be also clearly revealed. These results demonstrate that the K-kit developed in this study can be useful for observing living organisms and in situ monitoring bio-reaction.
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