“Using LCEM (liquid cell electron microscopy) it has been possible for the first time to capture early-stage nucleation events of organic molecular crystals used as APIs (active pharmaceutical ingredients),” said Dr Jennifer Cookman from the Bernal Institute at the University of Limerick and lead researcher for this study. “The importance of this method is that we can begin to understand how one crystal structure forms over another and, even more importantly, how these early-stage nucleation events manifest. We can also compare/contrast with classical nucleation theory and other crystal growth theories.”

The molecule studied, flufenamic acid, is a COX-inhibitor that acts as an anti-inflammatory. With increased study of this molecule, which is used in the pharmaceutical industry, researchers can potentially fine-tune its action as a medicinal drug and reduce its side-effects. What’s more, the molecular crystalline state is widely used across many industries; including electronics and agrochemicals. This research represents the first steps to analysing molecular crystal growth of not just flufenamic acid, but other molecules with implications for improvements in other industries.

The Ocean LPEM system’s unique ability to perform transmission electron microscopy in a liquid ethanol environment was essential for this research. Dr Cookman adds that by “using the DENSsolutions Ocean holder we were able to introduce an undersaturated liquid solution of the API to be visualised in the TEM protected from the high vacuum environment of the TEM.” Then crystal growth was induced by illuminating the sample with an electron beam which provided the energy needed to prompt the nucleation process.

In situ microscopy far exceeds previous ex situ observations as the team could produce live footage of each stage of crystal growth. Additionally, the in situ technique enabled the visualisation of the nucleation of flufenamic acid molecules in a working environment, a.k.a. ethanol. Performing this analysis in a liquid environment as opposed to a vacuum helped to meaningfully ascertain where and how different physical arrangements of crystal structures occur.

“This work brings focus to the use of electron microscopy and in particular in situ TEM equipment for characterisation,” added Dr Cookman. “That can be of utmost importance to the pharmaceutical industry and also in interim characterisation in pharmaceutical crystal research.”