Liquid-Phase Electron Microscopy Explained

Liquid-phase electron microscopy (LP EM) refers to a class of methods for imaging specimens in liquid with nanometer spatial resolution using electron microscopy. LP-EM overcomes the key limitation of electron microscopy: since the electron optics requires a high vacuum, the sample must be stable in a vacuum environment. Many types of specimens relevant to biology, materials science, chemistry, geology, and physics, however, change their properties when placed in a vacuum.

The ability to study liquid samples, particularly those involving water, with electron microscopy has been a wish ever since the early days of electron microscopy [1] but technical difficulties prevented early attempts from achieving high resolution.[2] Two basic approaches exist for imaging liquid specimens: i) closed systems, mostly referred to as liquid cell EM (LC EM), and ii) open systems, often referred to as environmental systems. In closed systems, thin windows made of materials such as silicon nitride or graphene are used to enclose a liquid for placement in the microscope vacuum. Closed cells have found widespread use in the past decade due to the availability of reliable window microfabrication technology.[3] [4] Graphene provides the thinnest possible window.[5] The oldest open system that gained widespread usage was environmental scanning electron microscopy (ESEM) of liquid samples on a cooled stage in a vacuum chamber containing a background pressure of vapor.[6] [7] Low vapor pressure liquids such as ionic liquids can also be studied in open systems.[8] LP-EM systems of both open and closed type have been developed for all three main types of electron microscopy, i.e., transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM), and scanning electron microscope (SEM).[9] Instruments integrating liquid-phase SEM with light microscopy have also been developed.[10] [11] Electron microscopic observation in liquid has been combined with other analytical methods such as electrochemical measurements [3] and energy-dispersive X-ray spectroscopy (EDX).[12]

The benefit of LP EM is the ability to study samples that do not withstand a vacuum or to study materials properties and reactions requiring liquid conditions. Examples of measurements enabled by this technique are the growth of metallic nanoparticles or structures in liquid,[13] [14] [15] [16] materials changes during the cycling of batteries,[8] [17] [18] electrochemical processes such as metal deposition,[3] dynamics of thin water films and diffusion processes,[19] biomineralization processes,[20] protein dynamics and structure,[21] [22] single-molecule localization of membrane proteins in mammalian cells,[4] [23] and the influence of drugs on receptors in cancer cells.[24]

The spatial resolution achievable can be in the sub-nanometer range and depends on the sample composition, structure and thickness, any window materials present, and the sensitivity of the sample to the electron dose required for imaging.[9] Nanometer resolution is obtained even in micrometers-thick water layers for STEM of nanomaterials of high atomic number.[4] [25] Brownian motion was found to be highly reduced with respect to a bulk liquid.[26] STEM detection is also possible in ESEM for imaging nanomaterials and biological cells in liquid.[27] [23] An important aspect of LP EM is the interaction of the electron beam with the sample [28] since the electron beam initiates a complex sequence of radiolytic reactions in water.[29] Nevertheless, quantitative analysis of LP EM data has yielded unique information in a range of scientific areas.[30] [31]

Notes and References

  1. Ruska, E. . 1942 . Beitrag zur uebermikroskopischen Abbildungen bei hoeheren Drucken . Kolloid Zeitschrift . 100 . 212–219 . 10.1007/bf01519549. 95628491 .
  2. Parsons. D.F. . Matricardi. V.R. . Moretz. R.C. . Turner. J.N. . 1974 . Electron microscopy and diffraction of wet unstained and unfixed biological objects . Advances in Biological and Medical Physics . 15 . 161–270 . 10.1016/B978-0-12-005215-8.50012-7 . 4135010 . 9780120052158 .
  3. Williamson. M.J.. Tromp. R.M.. Vereecken. P.M.. Hull. R.. Ross. F.M.. 2003 . Dynamic microscopy of nanoscale cluster growth at the solid-liquid interface . Nature Materials . 2 . 8. 532–536 . 10.1038/nmat944 . 12872162 . 2003NatMa...2..532W. 21379512.
  4. de Jonge. N.. Peckys. D.B.. Kremers. G.J.. Piston. D.W. . 2009 . Electron microscopy of whole cells in liquid with nanometer resolution . Proceedings of the National Academy of Sciences of the USA . 106 . 7. 2159–2164 . 10.1073/pnas.0809567106 . 2650183 . 19164524. 2009PNAS..106.2159J. free.
  5. Yuk. J.M.. etal . 2012 . High-resolution EM of colloidal nanocrystal growth using graphene liquid cells . Science . 336 . 6077. 61–64 . 10.1126/science.1217654 . 22491849. 2012Sci...336...61Y. 12984064.
  6. Danilatos. G.D.. Robinson. V.N.E.. 1979 . Principles of scanning electron microscopy at high specimen pressures . Scanning . 18 . 75–78 . 10.1002/sca.4950020202 . free .
  7. Book: Stokes. D.L. . 2008 . Principles and practice of variable pressure/environmental scanning electron microscopy (VP-SEM) . Wiley . Chichester, West-Sussex . 10.1002/9780470758731. 9780470758731 .
  8. Wang. C.M.. etal . 2010 . In situ transmission electron microscopy and spectroscopy studies of interfaces in Li ion batteries: challenges and opportunities . Journal of Materials Research . 25 . 8. 1541–1547 . 10.1557/jmr.2010.0198 . 2010JMatR..25.1541W.
  9. de Jonge. N.. Ross. F.M.. 2011 . Electron microscopy of specimens in liquid . Nature Nanotechnology . 6 . 11. 695–704 . 10.1038/nnano.2011.161 . 22020120. 2011NatNa...6..695D.
  10. Nishiyama. H.. etal . 2010 . Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film . J Struct Biol . 169 . 3. 438–449 . 10.1016/j.jsb.2010.01.005 . 20079847.
  11. Liv. N.. Lazic. I.. Kruit. P.. Hoogenboom. J.P. . 2014 . Scanning electron microscopy of individual nanoparticle bio-markers in liquid . Ultramicroscopy . 143 . 93–99 . 10.1016/j.ultramic.2013.09.002 . 24103705.
  12. Zaluzec. N.J.. Burke. M.G.. Haigh. S.J.. Kulzick. M.A. . 2014 . X-ray energy-dispersive spectrometry during in situ liquid cell studies using an analytical electron microscope . Microscopy and Microanalysis . 20 . 2. 323–329 . 10.1017/S1431927614000154 . 24564969. 2014MiMic..20..323Z. 32497871.
  13. Zheng. H.. etal . 2009 . Observation of single colloidal platinum nanocrystal growth trajectories . Science . 324 . 5932. 1309–1312 . 10.1126/science.1172104 . 19498166. 2009Sci...324.1309Z. 3731481.
  14. Donev. E.U.. Hastings. J.T. . 2009 . Electron-Beam-Induced Deposition of Platinum from a Liquid Precursor . Nano Letters . 9 . 7. 2715–2718 . 10.1021/nl9012216 . 19583284. 2009NanoL...9.2715D.
  15. Ahmad. N.. Wang. G.. Nelayah. J.. Ricolleau. C.. Alloyeau. D.. 2017 . Exploring the Formation of Symmetric Gold Nanostars by Liquid-Cell Transmission Electron Microscopy . Nano Lett . 17 . 7. 4194–4201 . 10.1021/acs.nanolett.7b01013 . 28628329. 2017NanoL..17.4194A.
  16. Song. B.. He. K.. Yuan. Y.. Sharifi-Asl. S.. Cheng. M.. Lu. J.. Saidi. W.. Shahbazian-Yassar. R.. 2018 . In situ study of nucleation and growth dynamics of Au nanoparticles on MoS2 nanoflakes . Nanoscale . 10 . 33. 15809–15818 . 10.1039/c8nr03519a . 30102314. 1472115.
  17. Hodnik. N.. Dehm. G.. Mayrhofer. K.J.J. . 2016 . Importance and Challenges of Electrochemical in Situ Liquid Cell Electron Microscopy for Energy Conversion Research . Accounts of Chemical Research . 49 . 9. 2015–2022 . 10.1021/acs.accounts.6b00330 . 27541965. free .
  18. Unocic. R.R.. etal . 2015 . Probing battery chemistry with liquid cell electron energy loss spectroscopy . Chemical Communications . 51 . 91. 16377–16380 . 10.1039/c5cc07180a . 26404766. 1237629.
  19. Mirsaidov. U.M.. Zheng. H.M.. Bhattacharya. D.. Casana. Y.. Matsudaira. P. . 2012 . Direct observation of stick-slip movements of water nanodroplets induced by an electron beam . Proceedings of the National Academy of Sciences of the USA . 109 . 19. 7187–7190 . 10.1073/pnas.1200457109 . 3358860 . 22517747. 2012PNAS..109.7187M. free.
  20. Smeets. P.J.. Cho. K.R.. Kempen. R.G.. Sommerdijk. N.A.. De Yoreo. J.J.. 2015 . Calcium carbonate nucleation driven by ion binding in a biomimetic matrix revealed by in situ electron microscopy . Nature Materials . 14 . 4. 394–399 . 10.1038/nmat4193 . 25622001. 2015NatMa..14..394S.
  21. Sugi. H.. etal. 1997 . Dynamic electron microscopy of ATP-induced myosin head movement in living muscle filaments . Proc. Natl. Acad. Sci. . 94 . 9. 4378–4392 . 10.1073/pnas.94.9.4378. 9113997. 20730. free .
  22. Mirsaidov. U.M.. Zheng. H.. Casana. Y.. Matsudaira. P. . 2012 . Imaging protein structure in water at 2.7 nm resolution by transmission electron microscopy . Biophysical Journal . 102 . 4. L15-7. 10.1016/j.bpj.2012.01.009 . 3283772 . 22385868. 2012BpJ...102L..15M.
  23. Peckys. D.B.. Korf. U.. de Jonge. N. . 2015 . Local variations of HER2 dimerization in breast cancer cells discovered by correlative fluorescence and liquid electron microscopy . Science Advances . 1 . 6. e1500165 . 10.1126/sciadv.1500165 . 4646781 . 26601217. 2015SciA....1E0165P.
  24. Peckys. D.B.. Korf. U.. Wiemann. S.. de Jonge. N.. 2017 . Liquid-phase electron microscopy of molecular drug response in breast cancer cells reveals irresponsive cell subpopulations related to lack of HER2 homodimers . Molecular Biology of the Cell . 3193–3202. 10.1091/mbc.E17-06-0381 . 28 . 23. 28794264 . 5687022.
  25. de Jonge. N.. Poirier-Demers. N.. Demers. H.. Peckys. D.B.. Drouin. D.. 2010 . Nanometer-resolution electron microscopy through micrometers-thick water layers . Ultramicroscopy . 110 . 9. 1114–1119 . 10.1016/j.ultramic.2010.04.001 . 2917648 . 20542380.
  26. Ring. E.A.. de Jonge. N. . 2012 . Video-frequency scanning transmission electron microscopy of moving gold nanoparticles in liquid . Micron . 43 . 11. 1078–1084 . 10.1016/j.micron.2012.01.010. 22386765. free.
  27. Bogner . A.. Thollet. G.. Basset. D.. Jouneau. P.H.. Gauthier. C. . 2005 . Wet STEM: A new development in environmental SEM for imaging nano-objects included in a liquid phase . Ultramicroscopy . 104 . 3–4. 290–301 . 10.1016/j.ultramic.2005.05.005. 15990230.
  28. Woehl. T.J.. etal . 2013 . Experimental procedures to mitigate electron beam induced artifacts during in situ fluid imaging of nanomaterials . Ultramicroscopy . 127 . 53–63 . 10.1016/j.ultramic.2012.07.018 . 22951261.
  29. Schneider. N.M.. etal. 2014 . Electron–water interactions and implications for liquid cell electron microscopy . Journal of Physical Chemistry C . 118 . 38. 22373–22382 . 10.1021/jp507400n .
  30. Book: Ross. F.M. . Frances M . Ross . 2017 . Liquid cell electron microscopy . Cambridge University Press . Cambridge. 10.1017/9781316337455 . 9781316337455 .
  31. Ross. F. M.. Wang. C.. de Jonge. N.. Transmission electron microscopy of specimens and processes in liquids. MRS Bulletin . 2016. 41. 10. 791–9. 10.1557/mrs.2016.212. 2016MRSBu..41..791R.

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