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Updated: Jan 29, 2026

Single Particle Cryo-Electron Microscopy: From Sample to Structure
Published on: May 29, 2021
Optimization of Infrared Microscopy to Assess Secondary Structure of Insulin Molecules Within Individual Subvisible
Malthe Møhl Schack1, Eva Horn Møller2, Anders V Friderichsen2
1Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark; Novo Nordisk A/S, Måløv, Denmark.
Abstract:
The analysis of subvisible particles is currently challenging but pivotal to the understanding and control of the quality of protein therapeutics. While a range of characterization methods is available for subvisible particles, information on the protein conformation in a particle-considered a possible parameter in eliciting unwanted immunogenicity of protein therapeutics-is especially challenging in the lower micrometer range using existing analytical technologies. Using 6 different protein particle populations, we show that transmission Fourier transform infrared (FTIR) microscopy can determine protein secondary structure in single particles down to 10 μm. The analytical setup presented here is able to immobilize protein particles and obtain transmission FTIR spectra on individual protein particles in their intact aqueous environment. Spectra of dried particles, on the other hand, were found to occasionally differ from spectra of particles in aqueous environment. In summary, using the analytical setup described in this study, transmission FTIR microscopy uniquely provides information on single protein particles in particle populations in their aqueous environment without interference from the background protein solution.
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