Quantitative analysis of a model pharmaceutical material, theophylline, by transmission electron microscopy
Authors: Cattle James , S'ari Mark , Abellán Patricia , Ramasse Quentin , Hondow Nicole , Brown Andy , Brydson Rik 
Published: December 2016

Modern electron microscopy (EM) techniques and hardware offer some of the highest attainable spatial resolutions for crystal imaging, making EM one of the best tools for microstructural analysis of a wide variety of materials. Organic materials, specifically pharmaceuticals, for which microstructure is a key part of their functionality would make ideal candidates for EM analysis, as it could provide useful feedback at various stages during drug development to check the presence of desired crystalline polymorphs, assess mixing quality, quantify crystal lattice defects and identify contamination. However, the major drawback to the use of EM is the high sensitivity of organic crystalline materials to electron beam exposure. Employing fluence rates that are typically used for inorganic samples would destroy all traces of crystallinity in most pharmaceutical materials[1]. Low‐dose techniques have been used for many years to analyse beam sensitive samples[2] using EM, but with recent improvements in CCD camera sensitivities, microscope stabilisation and current control, combined with existing low‐dose techniques, there is great opportunity for EM to be used for detailed organic materials analysis. Initially, the limits and flexibility of low‐dose techniques need to be tested and documented, both qualitatively and quantitatively. From this, a deeper understanding of the damage mechanisms at play can be drawn out, informing on the limits of organic crystalline materials analysis by EM and indicating appropriate damage mitigation strategies which could be employed in future studies.

Initial experiments with a model pharmaceutical, theophylline, observed by transmission electron microscopy (TEM) considered the effects of changing sample and electron beam conditions[3]. The aim was to determine the conditions which resulted in the highest critical dose (CD) (the dose where the intensity in a given diffraction spot decays to 1/e of its highest value). Since then, further experiments have been undertaken, including tests at 300 kV and a range of temperatures, from 93 K to 423 K. The ideal conditions identified use a high accelerating voltage with a graphene support for improved heat and electronic conduction, at a reduced sample temperature if necessary (only minor CD improvement in theophylline). Figure 1 shows the critical doses measured for the selection of variables investigated. Using this knowledge of theophylline's limits, lattice imaging has been attempted using a number of different techniques. Both bright field TEM and STEM imaging modes have been used and their results compared. Figure 2 shows a bright field STEM image of theophylline. Of note is that STEM results were acquired at a total electron fluence several times higher than the average CD of theophylline in TEM mode, suggesting an inherent damage reduction when analysing organic samples in STEM mode. Future work will focus on the use of improved hardware for direct lattice imaging in low‐dose TEM and determining the best conditions for STEM mode to exploit the potential differences between imaging modes.