10:45 AM - 12:15 PM
[MIS14-P01] Can living microorganisms be observed with a transmission electron microscope?
Keywords:TEM, in-situ observation, biomineralization
Recently, liquid samples have been successfully introduced into a transmission electron microscope (TEM) by using a liquid cell [1], and the observation technique has been established in the last decade. Various observations have been investigated and it has been found that even solutions with submicron thickness can be observed, and TEM observation of solution has been performed in many fields in recent years [e.g., 2,3]. Our group has also achieved results in cleaning of semiconductor surface, cement solidification process, agglomeration of amyloid-beta protein, crystallization of lysozyme protein, metal nanoparticles, photocatalytic particles, colloidal particles, salts and clay minerals [4-11]. Although it may seem that the technique can be applied directly to biological observation, it is very difficult because irradiating microorganisms with electron beams, the probe of TEM, induces cell death in an instant. Currently, microorganisms are only observed in a state of inactivation by freeze-drying or staining.
The bottleneck in TEM observation of processes involving organisms in real environments in water is the radiolysis of water and electron damage to the sample. When an aqueous solution is irradiated with electrons, radiolysis produces protons, ions, radicals, etc., and changes the pH. At a typical electron irradiation intensity (103-104 electron nm-2 s-1), for example, a solution with pH 10-11 will drop to about pH 7-8 [12]. The absorbed dose of the solution is also very large, about ~107 Gy s-1, and microorganisms die in microseconds to milliseconds, making observation of living cells difficult, and some papers have titled it probably impossible [13]. Therefore, even now that liquid-cell TEM has been established, cryo-TEM observation, in which samples are frozen before observation, is still widely used.
In cryo-TEM observation, samples are quickly frozen in liquid ethane and inserted into a TEM at low temperature and low electron dose. This technique allows observation of internal structures such as cells embedded in amorphous ice, but only in a dead state. The photographs are taken in a screening manner, looking for many individuals while taking care to avoid electron beam damage, and the original structural information is later restored by image processing [14]. Here, if the thickness of amorphous ice is reduced to obtain a clear image, the sample may be increasingly altered by drying. In atomic resolution observation, alteration due to drying cannot be ignored. Is it still impossible to observe live microorganisms by TEM?
Through our observations of lysozyme protein crystals, we feel that observation by liquid-cell TEM is less damaging to proteins than observation by cryo-TEM [6,8,11]. In addition, machine learning has enabled us to obtain clear images with nearly four orders of magnitude fewer electron beams than usual [15]. In this talk, we will discuss whether TEM can be used to observe live microorganisms for in situ TEM observation of biomineralization processes.
[1] M. J. Williamson et al. Nature Materials 2 (2003) 532.
[2] M. H. Nielsen et al. Science 345 (2014) 1158.
[3] F. M. Ross Science 350 (2015) aaa9886.
[4] Y. Sasaki, T. Yamazaki, Y. Kimura, ACS Applied Nano Materials, 5 (2022) 9495.
[5] Y. Kimura, H. Katsuno, T. Yamazaki, Faraday Discussions, 235 (2022) 81.
[6] Y. Kimura, Microscopy, 7 (2021) 13.
[7] Y. Igami, A. Tsuchiyama, T. Yamazaki, M. Matsumoto, Y. Kimura, Geochimica et Cosmochimica Acta 293 (2021) 86.
[8] T. Yamazaki, A. E. S. Van Driessche, Y. Kimura, Soft Matter, 16 (2020) 1955.
[9] K. Nakajima, T. Yamazaki, Y. Kimura, M. So, Y. Goto, H. Ogi, The Journal of Physical Chemistry Letters, 11 (2020) 6176.
[10] H. Satoh, Y. Kimura, E. Furukawa, Industrial & Engineering Chemistry Research, 57 (2018) 79.
[11] T. Yamazaki, Y. Kimura, P. G. Vekilov, E. Furukawa, M. Shirai, H. Matsumoto, A. E. S. Van Driessche, K. Tsukamoto, Proceedings of the National Academy of Sciences of the United States of America, 114 (2017) 2154.
[12] N. M. Schneider et al. J. Phys. Chem. C 118 (2014) 22373.
[13] N. de Jonge, D. B. Peckys, ACS Nano 10 (2016) 9061
[14] M. Adrian et al. Nature 308 (1984) 32.
[15] H. Katsuno, Y. Kimura, T. Yamazaki, I. Takigawa, Microscopy and Microanalysis, 28 (2022) 138
Acknowledgement
This work was supported by Grant-in-Aid for Scientific Research (S) of JSPS KAKENHI Grant Number 20H05657.
The bottleneck in TEM observation of processes involving organisms in real environments in water is the radiolysis of water and electron damage to the sample. When an aqueous solution is irradiated with electrons, radiolysis produces protons, ions, radicals, etc., and changes the pH. At a typical electron irradiation intensity (103-104 electron nm-2 s-1), for example, a solution with pH 10-11 will drop to about pH 7-8 [12]. The absorbed dose of the solution is also very large, about ~107 Gy s-1, and microorganisms die in microseconds to milliseconds, making observation of living cells difficult, and some papers have titled it probably impossible [13]. Therefore, even now that liquid-cell TEM has been established, cryo-TEM observation, in which samples are frozen before observation, is still widely used.
In cryo-TEM observation, samples are quickly frozen in liquid ethane and inserted into a TEM at low temperature and low electron dose. This technique allows observation of internal structures such as cells embedded in amorphous ice, but only in a dead state. The photographs are taken in a screening manner, looking for many individuals while taking care to avoid electron beam damage, and the original structural information is later restored by image processing [14]. Here, if the thickness of amorphous ice is reduced to obtain a clear image, the sample may be increasingly altered by drying. In atomic resolution observation, alteration due to drying cannot be ignored. Is it still impossible to observe live microorganisms by TEM?
Through our observations of lysozyme protein crystals, we feel that observation by liquid-cell TEM is less damaging to proteins than observation by cryo-TEM [6,8,11]. In addition, machine learning has enabled us to obtain clear images with nearly four orders of magnitude fewer electron beams than usual [15]. In this talk, we will discuss whether TEM can be used to observe live microorganisms for in situ TEM observation of biomineralization processes.
[1] M. J. Williamson et al. Nature Materials 2 (2003) 532.
[2] M. H. Nielsen et al. Science 345 (2014) 1158.
[3] F. M. Ross Science 350 (2015) aaa9886.
[4] Y. Sasaki, T. Yamazaki, Y. Kimura, ACS Applied Nano Materials, 5 (2022) 9495.
[5] Y. Kimura, H. Katsuno, T. Yamazaki, Faraday Discussions, 235 (2022) 81.
[6] Y. Kimura, Microscopy, 7 (2021) 13.
[7] Y. Igami, A. Tsuchiyama, T. Yamazaki, M. Matsumoto, Y. Kimura, Geochimica et Cosmochimica Acta 293 (2021) 86.
[8] T. Yamazaki, A. E. S. Van Driessche, Y. Kimura, Soft Matter, 16 (2020) 1955.
[9] K. Nakajima, T. Yamazaki, Y. Kimura, M. So, Y. Goto, H. Ogi, The Journal of Physical Chemistry Letters, 11 (2020) 6176.
[10] H. Satoh, Y. Kimura, E. Furukawa, Industrial & Engineering Chemistry Research, 57 (2018) 79.
[11] T. Yamazaki, Y. Kimura, P. G. Vekilov, E. Furukawa, M. Shirai, H. Matsumoto, A. E. S. Van Driessche, K. Tsukamoto, Proceedings of the National Academy of Sciences of the United States of America, 114 (2017) 2154.
[12] N. M. Schneider et al. J. Phys. Chem. C 118 (2014) 22373.
[13] N. de Jonge, D. B. Peckys, ACS Nano 10 (2016) 9061
[14] M. Adrian et al. Nature 308 (1984) 32.
[15] H. Katsuno, Y. Kimura, T. Yamazaki, I. Takigawa, Microscopy and Microanalysis, 28 (2022) 138
Acknowledgement
This work was supported by Grant-in-Aid for Scientific Research (S) of JSPS KAKENHI Grant Number 20H05657.