Imaging samples in liquids with electron microscopy can provide unique insights into biological systems, such as cells containing labelled proteins, and into processes of importance in materials science, such as nanoparticle synthesis and electrochemical deposition. Here we review recent progress in the use of electron microscopy in liquids and its applications. We examine the experimental challenges involved and the resolution that can be achieved with different forms of the technique. We conclude by assessing the potential role that electron microscopy of liquid samples can play in areas such as energy storage and bioimaging.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Nature Communications Open Access 23 June 2022
Nature Catalysis Open Access 30 December 2021
Dynamic transformation of cubic copper catalysts during CO2 electroreduction and its impact on catalytic selectivity
Nature Communications Open Access 18 November 2021
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ruska, E. Beitrag zur uebermikroskopischen Abbildungen bei hoeheren Drucken. Kolloid Z. 100, 212–219 (1942). This paper describes the first in situ TEM — its principle of operation is still the basis of most modern in situ TEM systems.
Thiberge, S. et al. Scanning electron microscopy of cells and tissues under fully hydrated conditions. Proc. Natl Acad. Sci. USA 101, 3346–3351 (2004). This paper made it clear that cells and tissue fully embedded in liquid can be imaged with a SEM with a resolution of several tens of nanometres.
Williamson, M. J., Tromp, R. M., Vereecken, P. M., Hull, R. & Ross, F. M. Dynamic microscopy of nanoscale cluster growth at the solid–liquid interface. Nature Mater. 2, 532–536 (2003). This paper describes the first use of a silicon nitride liquid cell for measuring a growth process in the TEM, and the first combination of electrical biasing with liquid cell microscopy to control and quantify a growth process.
de Jonge, N., Peckys, D. B., Kremers, G. J. & Piston, D. W. Electron microscopy of whole cells in liquid with nanometer resolution. Proc. Natl Acad. Sci. USA 106, 2159–2164 (2009). This paper is the first demonstration of the imaging of labelled proteins in whole eukaryotic cells in liquid with a resolution of several nanometres, using STEM.
Huang, J. Y. et al. In situ observation of the electrochemical lithiation of a single SnO2 nanowire electrode. Science 330, 1515–1520 (2010).
Zheng, H. et al. Observation of single colloidal platinum nanocrystal growth trajectories. Science 324, 1309–1312 (2009). This paper is the first demonstration of the study of nanoparticle growth in liquid with nanometre resolution in TEM.
Ruska, E. The development of the electron microscope and of electron microscopy. Nobel Lectures, Physics 1981–1990 (1986).
Reimer, L. & Kohl, H. Transmission Electron Microscopy: Physics of Image Formation (Springer, 2008).
Bozzola, J. J. & Russell, L. D. Electron Microscopy (Jones and Bartlett, 1992).
von Ardenne, M. Ueber eun 200 kV-Universal-Elektronenmikroskop mit Objektabschattungsvorrichtung. Z. Phys. 117, 657–688 (1941).
Donnelly, S. E. et al. Ordering in a fluid inert gas confined by flat surfaces. Science 296, 507–510 (2002).
Parsons, D. F., Matricardi, V. R., Moretz, R. C. & Turner, J. N. Electron microscopy and diffraction of wet unstained and unfixed biological objects. Adv. Biol. Med. Phys. 15, 161–270 (1974).
Helveg, S. et al. Atomic-scale imaging of carbon nanofibre growth. Nature 427, 426–429 (2004).
Dai, L. L., Sharma, R. & Wu, C. Y. Self-assembled structure of nanoparticles at a liquid–liquid interface. Langmuir 21, 2641–2643 (2005).
Danilatos, G. D. & Robinson, V. N. E. Principles of scanning electron microscopy at high specimen pressures. Scanning 18, 75–78 (1979).
Stokes, D. J. Recent advances in electron imaging, image interpretation and applications: environmental scanning electron microscopy. Phil. Trans. R. Soc. Lond. A 361, 2771–2787 (2003).
Stokes, D. L. Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP-SEM) (Wiley, 2008).
Abrams, I. M. & McBain, J. W. A closed cell for electron microscopy. J Appl. Phys. 15, 607–609 (1944).
Daulton, T. L., Little, B. J., Lowe, K. & Jones-Meehan, J. In situ environmental cell–transmission electron microscopy study of microbial reduction of chromium(VI) using electron energy loss spectroscopy. Microsc. Microanal. 7, 470–485 (2001).
Nishijima, K., Yamasaki, J., Orihara, H. & Tanaka, N. Development of microcapsules for electron microscopy and their application to dynamical observation of liquid crystals in transmission electron microscopy. Nanotechnology 15, S329–S332 (2004).
Mohanty, N., Fahrenholtz, M., Nagaraja, A., Boyle, D. & Berry, V. Impermeable graphenic encasement of bacteria. Nano Lett. 11, 1270–1275 (2011).
Grogan, J. M. & Bau, H. H. The nanoaquarium: a platform for in situ transmission electron microscopy in liquid media. J. Microelectromech. Syst. 19, 885–894 (2010).
Franks, R. et al. A study of nanomaterial dispersion in solution by wet-cell transmission electron microscopy. J. Nanosci. Nanotechnol. 8, 4404–4407 (2008).
Liu, K. L. et al. Novel microchip for in situ TEM imaging of living organisms and bio-reactions in aqueous conditions. Lab Chip 8, 1915–1921 (2008).
Ring, E. A. & de Jonge, N. Microfluidic system for transmission electron microscopy. Microsc. Microanal. 16, 622–629 (2010).
de Jonge, N., Poirier-Demers, N., Demers, H., Peckys, D. B. & Drouin, D. Nanometer-resolution electron microscopy through micrometers-thick water layers. Ultramicroscopy 110, 1114–1119 (2010).
Peckys, D. B., Veith, G. M., Joy, D. C. & de Jonge, N. Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope. PLoS One 4, e8214 (2009).
Creemer, J. F. et al. Atomic-scale electron microscopy at ambient pressure. Ultramicroscopy 108, 993–998 (2008). This paper describes advances in high-pressure cells for the TEM that combine enhanced functionality with the ability to image at atomic resolution.
Creemer, J. F. et al. A MEMS reactor for atomic-scale microscopy of nanomaterials under industrially relevant conditions. J. Microelectromech. Syst. 19, 254–264 (2010).
Kawasaki, T., Ueda, K., Ichihashi, M. & Tanji, T. Improvement of windowed type environmental-cell transmission electron microscope for in situ observation of gas-solid interactions. Rev. Sci. Instr. 80, 113701–113705 (2009).
Klein, K. L., Anderson, I. M. & de Jonge, N. Transmission electron microscopy with a liquid flow cell. J. Microsc. 242, 117–123 (2011).
Zheng, H., Claridge, S. A., Minor, A. M., Alivisatos, A. P. & Dahmen, U. Nanocrystal diffusion in a liquid thin film observed by in situ transmission electron microscopy. Nano Lett. 9, 2460–2465 (2009).
Nishiyama, H. et al. Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film. J. Struct. Biol. 169, 438–449 (2010).
Inami, W., Nakajima, K., Miyakawa, A. & Kawata, Y. Electron beam excitation assisted optical microscope with ultra-high resolution. Opt. Express 18, 12897–12902 (2010).
Evans, J. E., Jungjohann, K. L., Browning, N. D. & Arslan, I. Controlled growth of nanoparticles from solution with in situ liquid transmission electron microscopy. Nano Lett. 11, 2809–2813 (2011).
Sali, A., Glaeser, R., Earnest, T. & Baumeister, W. From words to literature in structural proteomics. Nature 422, 216–225 (2003).
Stahlberg, H. & Walz, T. Molecular electron microscopy: state of the art and current challenges. ACS Chem. Biol. 3, 268–281 (2008).
Pierson, J., Sani, M., Tomova, C., Godsave, S. & Peters, P. J. Toward visualization of nanomachines in their native cellular environment. Histochem. Cell. Biol. 132, 253–262 (2009).
Kirk, S. E., Skepper, J. N. & Donald, A. M. Application of environmental scanning electron microscopy to determine biological surface structure. J. Microsc. 233, 205–224 (2009).
Collins, S. P. et al. Advantages of environmental scanning electron microscopy in studies of microorganisms. Microsc. Res. Techniq. 25, 398–405 (1993).
Bogner, A., Thollet, G., Basset, D., Jouneau, P. H. & Gauthier, C. Wet STEM: A new development in environmental SEM for imaging nano-objects included in a liquid phase. Ultramicroscopy 104, 290–301 (2005).
Xiao, Y., Patolsky, F., Katz, E., Hainfeld, J. F. & Willner, I. 'Plugging into enzymes': nanowiring of redox enzymes by a gold nanoparticle. Science 299, 1877–1881 (2003).
Barshack, I. et al. A novel method for 'wet' SEM. Ultrastruct. Pathol. 28, 29–31 (2004).
Melo, R. C., Sabban, A. & Weller, P. F. Leukocyte lipid bodies: inflammation-related organelles are rapidly detected by wet scanning electron microscopy. J. Lipid. Res. 47, 2589–2594 (2006).
Sugi, H. et al. Dynamic electron microscopy of ATP-induced myosin head movement in living muscle filaments. Proc. Natl Acad. Sci. USA 94, 4378–4392 (1997).
Matricardi, V. R., Moretz, R. C. & Parsons, D. F. Electron diffraction of wet proteins: catalase. Science 177, 268–270 (1972).
Lippincott-Schwartz, J. & Manley, S. Putting super-resolution fluorescence microscopy to work. Nature Methods 6, 21–23 (2009).
Peckys, D. B. & de Jonge, N. Visualization of gold nanoparticle uptake in living cells with liquid scanning transmission electron microscopy. Nano Lett. 11, 1733–1738 (2011).
Peckys, D. B., Mazur, P., Gould, K. L. & de Jonge, N. Fully hydrated yeast cells imaged with electron microscopy. Biophys. J. 100, 2522–2529 (2011).
Murai, T. et al. Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration. J. Biol. Chem. 286, 1999–2007 (2011).
Dukes, M. J., Peckys, D. B. & de Jonge, N. Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid. ACS Nano 4, 4110–4116 (2010).
Radisic, A., Vereecken, P. M., Hannon, J. B., Searson, P. C. & Ross, F. M. Quantifying electrochemical nucleation and growth of nanoscale clusters using real-time kinetic data. Nano Lett. 6, 238–242 (2006).
Scharifker, B. R. & Hills, G. J. Theoretical and experimental studies of multiple nucleation. Electrochim. Acta 28, 879–889 (1983).
Radisic, A., Ross, F. M. & Searson, P. C. In situ study of the growth kinetics of individual island electrodeposition of copper. J. Phys. Chem. B 110, 7862–7868 (2006).
Radisic, A., Vereecken, P. M., Searson, P. C. & Ross, F. M. The morphology and nucleation kinetics of copper islands during electrodeposition. Surf. Sci. 600, 1817–1826 (2006).
Wise, M. E., Biskos, G., Martin, S. T., Russell, L. M. & Buseck, P. R. Phase transitions of single salt particles studied using a transmission electron microscope with an environmental cell. Aerosol. Sci. Tech. 39, 849–856 (2005).
Gai, P. L. Development of wet environmental TEM (wet-ETEM) for in situ studies of liquid-catalyst reactions on the nanoscale. Microsc. Microanal. 8, 21–28 (2002). The first observation by TEM of an industrially important liquid-phase catalytic reaction.
Gai, P. L. & Harmer, M. A. Surface atomic defect structures and growth of Au nanorods. Nano Lett. 2, 771–774 (2002).
Gabrisch, H., Kjeldgaard, L., Johnson, E. & Dahmen, U. Equilibrium shape and interface roughening of small liquid Pb inclusions in solid Al. Acta Mater. 49, 4259–4269 (2001).
Ross, F. M., Tersoff, J. & Reuter, M. C. Sawtooth faceting in silicon nanowires. Phys. Rev. Lett. 95, 146104 (2005).
Eswaramoorthy, S. K., Howe, J. M. & Muralidharan, G. In situ determination of the nanoscale chemistry and behavior of solid-liquid systems. Science 318, 1437–1440 (2007).
Lee, J. G. & Mori, H. In situ observation of alloy phase formation in nanometre-sized particles in the Sn–Bi system. Philos. Mag. 84, 2675–2686 (2004).
Howe, J. M. & Saka, H. In situ transmission electron microscopy studies of the solid–liquid interface. MRS Bull. 29, 951–957 (2004).
Kuwabata, S., Kongkanand, A., Oyamatsu, D. & Torimoto, T. Observation of ionic liquid by scanning electron microscope. Chem. Lett. 35, 600–601 (2006).
Roy, P., Lynch, R. & Schmuki, P. Electron beam induced in vacuo Ag deposition on TiO2 from ionic liquids. Electrochem. Comm. 11, 1567–1570 (2009).
Joy, D. C. & Joy, C. S. Scanning electron microscope imaging in liquids — some data on electron interactions in water. J. Microsc. 221, 84–99 (2005).
Hyun, J. K., Ercius, P. & Muller, D. A. Beam spreading and spatial resolution in thick organic specimens. Ultramicroscopy 109, 1–7 (2008).
Loos, J. et al. Electron tomography on micrometer-thick specimens with nanometer resolution. Nano Lett. 9, 1704–1708 (2009).
Demers, H., Poirier-Demers, N., Drouin, D. & de Jonge, N. Simulating STEM imaging of nanoparticles in micrometers-thick substrates. Microsc. Microanal. 16, 795–804 (2010).
Sousa, A. A., Hohmann-Marriott, M. F., Zhang, G. & Leapman, R. D. Monte Carlo electron-trajectory simulations in bright-field and dark-field STEM: implications for tomography of thick biological sections. Ultramicroscopy 109, 213–221 (2009).
Spence, J. C. H. High-Resolution Electron Microscopy (Oxford Univ. Press, 2003).
Hohmann-Marriott, M. F. et al. Nanoscale 3D cellular imaging by axial scanning transmission electron tomography. Nature Methods 6, 729–731 (2009).
Aoyama, K., Takagi, T., Hirase, A. & Miyazawa, A. STEM tomography for thick biological specimens. Ultramicroscopy 109, 70–80 (2008).
Crewe, A. V., Wall, J. & Langmore, J. Visibility of single atoms. Science 168, 1338–1340 (1970).
Krivanek, O. L. et al. Atom-by-atom structural and chemical analysis by annular dark-field electron microscopy. Nature 464, 571–574 (2010).
Goldstein, J. I. in Introduction to Analytical Electron Microscopy (eds Hren, J. J., Goldstein, J. I. & Joy, D. C.) 83–120 (Plenum Press, 1979).
Thiberge, S., Zik, O. & Moses, E. An apparatus for imaging liquids, cells, and other wet samples in the scanning electron microscopy. Rev. Sci. Instrum. 75, 2280–2289 (2004).
Fenter, P., Lee, S. S., Zhang, Z. & Sturchio, N. C. In situ imaging of orthoclase-aqueous solution interfaces with X-ray reflection interface microscopy. J. Appl. Phys. (in the press).
Garrett, B. C. et al. Role of water in electron-initiated processes and radical chemistry: issues and scientific advances. Chem. Rev. 105, 355–390 (2005).
Donev, E. U., Schardein, G., Wright, J. C. & Hastings, J. T. Substrate effects on the electron-beam-induced deposition of platinum from a liquid precursor. Nanoscale 3, 2709–2717 (2011).
Hui, S. W. & Parsons, D. F. Electron diffraction of wet biological membranes. Science 184, 77–78 (1974).
Kenworthy, A. K. et al. Dynamics of putative raft-associated proteins at the cell surface. J. Cell Biol. 165, 735–746 (2004).
Holmqvist, P., Dhont, J. K. G. & Lang, P. R. Anisotropy of Brownian motion caused only by hydrodynamic interaction with a wall. Phys. Rev. E 74, 021402 (2006).
Pawley, J. B. Handbook of Biological Confocal Microscopy (Springer, 1995).
Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007). This paper reviews several light microscopy approaches to break the diffraction barrier.
Betzig, E., Trautman, J. K., Harris, T. D., Weiner, J. S. & Kostelak, R. L. Breaking the diffraction barrier: optical microscopy on a nanometric scale. Science 251, 1468–1470 (1991).
Chao, W., Harteneck, B. D., Liddle, J. A., Anderson, E. H. & Attwood, D. T. Soft X-ray microscopy at a spatial resolution better than 15 nm. Nature 435, 1210–1213 (2005).
Larabell, C. A. & Nugent, K. A. Imaging cellular architecture with X-rays. Curr. Opin. Struct. Biol. 20, 623–631 (2010).
Muller, D. J., Helenius, J., Alsteens, D. & Dufrene, Y. F. Force probing surfaces of living cells to molecular resolution. Nature Chem. Biol. 5, 383–390 (2009).
Allison, D. P., Mortensen, N. P., Sullivan, C. J. & Doktycz, M. J. Atomic force microscopy of biological samples. Nanomed. Nanobiotechnol. 2, 618–634 (2010).
Fleming, A. J., Kenton, B. J. & Leang, K. K. Bridging the gap between conventional and video-speed scanning probe microscopes. Ultramicroscopy 110, 1205–1214 (2010).
Sulchek, T. et al. High-speed atomic force microscopy in liquid. Rev. Sci. Instrum. 71, 2097–2099 (2000).
Langmore, J. P. & Smith, M. F. Quantitative energy-filtered electron microscopy of biological molecules in ice. Ultramicroscopy 46, 349–373 (1992).
Haider, M., Hartel, P., Muller, H., Uhlemann, S. & Zach, J. Current and future aberration correctors for the improvement of resolution in electron microscopy. Phil. Trans. R. Soc. A 367, 3665–3682 (2009).
Flannigan, D. J., Barwick, B. & Zewail, A. H. Biological imaging with 4D ultrafast electron microscopy. Proc. Natl Acad. Sci. USA 107, 9933–9937 (2010).
Campbell, G. H., LaGrange, T., Kim, J. S., Reed, B. W. & Browning, N. D. Quantifying transient states in materials with the dynamic transmission electron microscope. J. Electron Microsc. 59 (suppl. 1), S67–S74 (2010).
Kruit, P. & Jansen, G. H. in Handbook of Charged Particle Optics (ed. Orloff, J.) 275–318 (CRC Press, 1997).
Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nature Rev. Mol. Cell Biol. 2, 444–456 (2001).
Agronskaia, A. V. et al. Integrated fluorescence and transmission electron microscopy. J. Struct. Biol. 164, 183–189 (2008).
Shu, X. et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).
Chou, L. Y., Ming, K. & Chan, W. C. Strategies for the intracellular delivery of nanoparticles. Chem. Soc. Rev. 40, 233–245 (2011).
Tkachenko, A. G. et al. Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains. Bioconjugate Chem. 15, 482–490 (2004).
Tantra, R. & Knight, A. Cellular uptake and intracellular fate of engineered nanoparticles: A review on the application of imaging techniques. Nanotoxicology 5, 381–392 (2010).
Ross, F. M. Electrochemical nucleation, growth and dendrite formation in liquid cell TEM. Microsc. Microanal. 16, S326–S327 (2010).
Wang, C. M. et al. In situ transmission electron microscopy and spectroscopy studies of interfaces in Li-ion batteries: challenges and opportunities. J. Mater. Res. 25, 1541–1547 (2010).
This work was supported by Vanderbilt University School of Medicine and by IBM.
The authors declare no competing financial interests.
About this article
Cite this article
de Jonge, N., Ross, F. Electron microscopy of specimens in liquid. Nature Nanotech 6, 695–704 (2011). https://doi.org/10.1038/nnano.2011.161
This article is cited by
Nature Communications (2022)
Nature Catalysis (2022)
Nanoscopic and in-situ cross-sectional observations of Li-based conversion coating formation using liquid-phase TEM
npj Materials Degradation (2021)
Thermoresponsive polymer assemblies via variable temperature liquid-phase transmission electron microscopy and small angle X-ray scattering
Nature Communications (2021)