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Suitability of a liquid TEM cell to study calcium phosphate nucleation

Here, we use and assess an in-situ liquid TEM cell to study solution-phase self-assembly of calcium phosphate nanoaggregates where nucleation is driven by dissolution of a bioactive glass. We aim to increase understanding of solution-phase nucleation mechanisms of biomaterials. And how this can be applied to the mineralisation of organic self-assembly macromolecules, the design and construction of smart materials for dental, orthopaedic, sensing and drug delivery applications, etc.


Elkin Lopez-Fontal


Dr Richard Langford


Glaxo Smith Kline (GSK)

The in-situ imaging system is formed by a liquid cell which consists of a top chip and spacer bottom chip that stack together forming an environmental cell (Figure 1). The chips have a small window made of silicon nitrate that allows for the electron beam to go through and image what’s happening in the environmental cell.


TEM liquid cell

Figure 1: Assembled in-situ TEM Liquid Cell.



Living organisms remarkably integrate organic macromolecules and inorganic minerals to form well sound structures like shells, bones, teeth, etc.1, 2 Inorganic materials are hard and stiff but brittle. In contrast, organic ones are relatively soft and pliable but tough.1, 2 So, Nature, by combining inorganic and organic materials has designed sophisticated biocomposites with particular and well defined mechanical properties.3

An increase in the understanding of biomineralisation processes is of large interest to the scientific community. Particularly, the study of bioglasses and their role in calcium phosphate nucleation and crystallization pathway of hydroxyapatite are still under debate and intensive research.


Electron microscopy is a widespread technique to image nanoparticles of all types. However, since soft matter is usually constituted by low atomic number elements (Z < 16) the contrast expected is very low and depending on materials it can be no discernible at all. To solve this problem, usually, soft-matter samples are stained with heavier elements (metals or halides) which in general allow better imaging but sometimes can induce charging causing damage to the soft material. Cryo-transmission electron microscopy can be a better answer as the sample is rapidly frozen in time and imaged which by and large is considered a good approximation to the material’s native solution-phase state. However, it would not be applicable, for instance, for real-time observations of particles dynamics.4

In-situ electron microscopy is a technique used for liquid-phase systems in which liquid-phase and liquid-solid interfacial processes are an intrinsic part.5 And where the motion of nanomaterials in their native environment can be observed: calcium carbonate nucleation,5 self-assembly of octopod shaped nanocrystals,6 motion of micellar nanoparticles,7 nanoparticle diffusion and self-Assembly.8, 9

In collaboration with GSK, the Cavendish laboratory has taken delivery of an in-situ TEM liquid cell holder aiming to assess its suitability to study biomineralisation processes.

Some preliminary results

Figure 2 shows stills of an in-situ calcium phosphate nucleation. Round amorphous calcium phosphate particles formed onto which nucleated a network of branches. Extensive nucleation and dissolution around the branches were visible as a function of time which lead to the formation of larger aggregates. 

BF TEM images

Figure 2: time series in-situ BF TEM images showing in-situ calcium phosphate nucleation. Red circles show nuclei arriving or sometimes leaving the big aggregate.



  1. Olszta, M. J., et al., Bone structure and formation: A new perspective. Materials Science and Engineering: R: Reports 2007, 58 (3-5), 77-116.
  2. Mann, S., Biomineralization: the form(id)able part of bioinorganic chemistry! Journal of the Chemical Society-Dalton Transactions 1997, (21), 3953-3961.
  3. Mann, S., Biomineralization: principles and concepts in bioinorganic materials chemistry. Oxford University Press on Demand: 2001; Vol. 5.
  4. de Jonge, N.; Ross, F. M., Electron microscopy of specimens in liquid. Nature nanotechnology 2011, 6 (11), 695-704.
  5. Nielsen, M. H., et al., Liquid Cell TEM for Studying Environmental and Biological Mineral Systems. 2017, 316-333.
  6. Sutter, E., et al., In situ microscopy of the self-assembly of branched nanocrystals in solution. Nature communications 2016, 7, 11213.
  7. Proetto, M. T., et al., Dynamics of soft nanomaterials captured by transmission electron microscopy in liquid water. J Am Chem Soc 2014, 136 (4), 1162-5.
  8. Demmert, A. C., et al., Visualizing Macromolecules in Liquid at the Nanoscale. 2017, 356-370.
  9. Woehl, T. J.; Prozorov, T., The Mechanisms for Nanoparticle Surface Diffusion and Chain Self-Assembly Determined from Real-Time Nanoscale Kinetics in Liquid. The Journal of Physical Chemistry C 2015, 119 (36), 21261-21269.
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