Hostname: page-component-78c5997874-s2hrs Total loading time: 0 Render date: 2024-11-13T01:54:56.251Z Has data issue: false hasContentIssue false
  • English
  • Français

In Situ Study of Live Specimens in an Environmental Scanning Electron Microscope

Published online by Cambridge University Press:  02 May 2013

Eva Tihlaříková ,
Vilém Neděla  and
Makoto Shiojiri
Eva Tihlaříková
Affiliation:
Institute of Scientific Instruments of the ASCR, v.v.i., Královopolská 147, Brno 612 64, Czech Republic
Vilém Neděla*
Affiliation:
Institute of Scientific Instruments of the ASCR, v.v.i., Královopolská 147, Brno 612 64, Czech Republic
Makoto Shiojiri
Affiliation:
Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
*
*Corresponding author.vilem@isibrno.cz
Get access

Abstract

In this paper we introduce new methodology for the observation of living biological samples in an environmental scanning electron microscope (ESEM). The methodology is based on an unconventional initiation procedure for ESEM chamber pumping, free from purge–flood cycles, and on the ability to control thermodynamic processes close to the sample. The gradual and gentle change of the working environment from air to water vapor enables the study of not only living samples in dynamic in situ experiments and their manifestation of life (sample walking) but also its experimentally stimulated physiological reactions. Moreover, Monte Carlo simulations of primary electron beam energy losses in a water layer on the sample surface were studied; consequently, the influence of the water thickness on radiation, temperature, or chemical damage of the sample was considered.

Keywords

live biological samplehigh-pressure environmenthydration systemin situ observationMonte Carlo simulationmethodology
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 4 , August 2013 , pp. 914 - 918
Copyright
Copyright © Microscopy Society of America 2013 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

These authors contributed equally to this work.

References

Autrata, R., Schauer, P., Kvapil, J. & Kvapil, J. (1978). A single crystal of YAG—New fast scintillator in SEM, J Phys E: Sci Instrum 11, 707–708.CrossRefGoogle Scholar
Bozzola, J.J. & Russell, L.D. (1999). Electron Microscopy: Principles and Techniques for Biologists. Sudbury, MA, USA: Jones & Bartlett Learning.Google Scholar
Cameron, R.E. & Donald, A.M. (1994). Minimizing sample evaporation in the environmental scanning electron microscope. J Microsc 173, 227–237.Google Scholar
Danilatos, G.D. (1981a). The examination of fresh or living plant material in an environmental scanning electron microscope. J Microsc 121, 235–238.CrossRefGoogle Scholar
Danilatos, G.D. (1981b). Design and construction of an atmospheric or environmental SEM (part 1). Scanning 4, 9–20.Google Scholar
Danilatos, G.D. (1988). Foundations of Environmental Scanning Electron Microscopy. Sydney, Australia: Academic Press.CrossRefGoogle Scholar
Danilatos, G.D. (2012). Velocity and ejector-jet assisted differential pumping: Novel design stages for environmental SEM. Micron 43, 600–611.CrossRefGoogle Scholar
Danilatos, G.D. & Postle, R. (1982). The environmental scanning electron microscope and its applications. Scan Electron Microsc 1982, 1–16.Google Scholar
Danilatos, G.D. & Robinson, V.N.E. (1979). Principles of scanning electron microscopy at high specimen pressures. Scanning 2, 72–82.Google Scholar
De Jonge, N. & Ross, F.M. (2011). Electron microscopy of specimens in liquid. Nat Nanotech 6, 695–704.CrossRefGoogle ScholarPubMed
Donald, A.M. (2003). The use of environmental scanning electron microscopy for imaging wet and insulating materials. Nat Mater 2, 511–516.CrossRefGoogle ScholarPubMed
Everhart, T.E. & Thornley, R.F.M. (1960). Wide-band detector for micro-microampere low-energy electron currents, J Sci Instrum 37, 246–248.CrossRefGoogle Scholar
Fukushima, K., Ishikawa, A. & Fukami, A. (1985). Injection of liquid into environmental cell for in situ observations. J Electron Microsc 34, 47–51.Google Scholar
Hashimoto, H., Naiki, T., Eto, T. & Fujiwara, K. (1968). High temperature gas reaction specimen chamber for an electron microscope. Jpn J Appl Phys 7, 946–953.Google Scholar
Hayat, M.A. (2000). Principles and Techniques of Electron Microscopy: Biological Applications. UK: Cambridge University Press.Google Scholar
Ivanchenko, V., Apostolakis, J., Bagulya, A., Abdelouahed, H.B., Black, R., Bogdanov, A., Burkhard, H., Chauvie, S., Cirrone, P., Cuttone, G., Depaola, G., Di Rosa, F., Elles, S., Francis, Z., Grichine, V., Gumplinger, P., Gueye, P., Incerti, S., Ivanchenko, A., Jacquemier, J., Lechner, A., Longo, F., Kadri, O., Karakatsanis, N., Karamitros, M., Kokoulin, R., Kurashige, H., Maire, M., Mantero, A., Mascialino, B., Moscicki, J., Pandola, L., Perl, J., Petrovic, I., Ristic-Fira, A., Romano, F., Russo, G., Santin, G., Schaelicke, A., Toshito, T., Tran, H., Urban, L., Yamashita, T. & Zacharatou, C. (2011). Recent improvement in Geant4 electromagnetic physics models and interfaces. J Nucl Sci Technol 2, 898–903.Google Scholar
Jirák, J., Neděla, V., Černoch, P., Čudek, P. & Runštuk, J. (2010). Scintillation SE detector for variable pressure scanning electron microscopes. J Microsc 239, 233–238.CrossRefGoogle ScholarPubMed
Lane, W.C. (1970). The environmental control stage. Scan Electron Microsc 1970, 43–48.Google Scholar
McGregor, J.E. & Donald, A.M. (2010). ESEM imaging of dynamic biological processes: The closure of stomatal pores. J Microsc 239, 135–141.Google Scholar
Neděla, V. (2007). Methods for additive hydration allowing observation of fully hydrated state of wet samples in environmental SEM. Microsc Res Tech 70, 95–100.CrossRefGoogle ScholarPubMed
Neděla, V. (2010). Controlled dehydration of a biological sample using an alternative form of environmental SEM. J Microsc 237, 7–11.Google Scholar
Neděla, V. & Jirák, J. (2012). Ionisation detector for environmental scanning electron microscope. Patent EP 2195822, Czech Patent No. 299864. Google Scholar
Neděla, V., Konvalina, I., Lencová, B. & Zlámal, J. (2011). Comparison of calculated, simulated and measured signal amplification in a variable pressure SEM. Nucl Instrum Methods Phys Res A 645, 79–83.Google Scholar
Peckys, D.B., Mazur, P., Gould, K.L. & Jonge, N. (2011). Fully hydrated yeast cells imaged with electron microscopy. Biophys J 100, 2522–2539.CrossRefGoogle ScholarPubMed
Robards, A.W. & Wilson, A.J. (1993). Procedures in Electron Microscopy. Chichester, UK: Wiley.Google Scholar
Robinson, V.N.E. (1975). A wet stage modification to a scanning electron microscope. J Microsc 103, 71–77.Google Scholar
Roos, N. & Morgan, A.J. (1990). Cryopreparation of Thin Biological Specimens for Electron Microscopy. Oxford, UK: Oxford Science Publications.Google Scholar
Royall, C.P., Thiel, B.L. & Donald, A.M. (2001). Radiation damage of water in environmental scanning electron microscopy. J Microsc 204, 185–195.Google Scholar
Ruska, E. (1942). Beitrag zur übermikroskopischen Abbildung bei höheren Drucken. Kolloid 100, 212–219.Google Scholar
Stokes, D.J. (2003). Investigating biological ultrastructure using environmental scanning electron microscopy (ESEM). In Science, Technology and Education of Microscopy an Overview, Méndez-Vilas, A. (Ed.), pp. 564–570. Spain: Formatex Research Center.Google Scholar
Stokes, D.J. (2006). Progress in the study of biological specimens using ESEM. Infocus 2, 64–72.Google Scholar

Tihlaříková et al. supplementary movie

Movie

Download Tihlaříková et al. supplementary movie(Video)
Video 1.5 MB