Scientific Programme

This year our Plenary Speakers are Professor Ian Gilmore (National Physical Laboratory, UK) who pioneered OrbiSIMS technology, revolutionising high-resolution molecular imaging for biomedical and material sciences and Dr Carla Newman (GlaxoSmithKline, UK) who is a leading expert in applying single cell mass spectrometry in pharmaceutical research and drug discovery.

Keynote Abstracts

Dr Carla Newman (GlaxoSmithKline, UK)

Single-cell analysis in drug discovery: enhancing granularity

Akshada Gajbhiye¹, Lennart Huizing¹, and Carla Newman¹

Cellular and Imaging Dynamics, IVIVT, GlaxoSmithKline, Stevenage, United Kingdom

Understanding cellular heterogeneity is critical for advancing pharmaceutical research, as subtle variations at the cellular level can profoundly impact drug efficacy and safety. While advanced microscopy techniques provide valuable insights into cellular morphology and dynamics, making them indispensable for identifying phenotypic variabilities, these methods alone cannot unravel the intricate molecular mechanisms that govern biological processes. In particular, the pharmaceutical industry's move toward more precise therapeutics necessitates a deeper characterization of cellular responses that can only be achieved by integrating proteomic analyses.

In this study, we explore the molecular basis of amiodarone-induced phospholipidosis, a condition marked by the accumulation of phospholipids within the endosomal-lysosomal system, leading to lysosomal dysfunction and lipid storage disorders. Traditional imaging identified by fluorescence labeling, including the use of Lysotracker, enabled the initial detection and visual confirmation of cellular alterations. These imaging modalities were crucial for selecting cells that exhibit aberrant lysosomal morphology, thereby pinpointing the cells most affected by the drug.

To gain a comprehensive understanding of the biological pathways involved, we employed Single Live-Cell Mass Spectrometry (SiLC-MS) using the tims-TOF Ultra system, a technology capable of capturing minute protein quantities in single cells. Precise cell sorting was achieved with the CellenONE single-cell dispenser, ensuring that isolated macrophages—selected based on cell size and fluorescence signals—were optimally prepared for proteomic analysis. The subsequent protein extraction and digestion were carried out in micro-scale conditions to maintain the integrity of the single-cell proteome.

Proteomic data acquisition was conducted in Data-Independent Acquisition (DIA) mode through coupling the tims-TOF Ultra system with a nano-elute Liquid Chromatography system. Data interpretation was facilitated using Spectronaut Software, which enabled the systematic quantification of protein expression levels and pathway activation states within individual cells. The integration of high-resolution imaging with in-depth single-cell proteomics offers unprecedented insight into the heterogeneity of drug-induced cellular responses, providing a molecular fingerprint of lysosomal impairment.

The findings from this study underscore the importance of combining microscopy and proteomics to reveal the underlying biological processes that contribute to cell heterogeneity. Such detailed characterization is not only essential for understanding the mechanistic basis of drug-induced lysosomal dysfunction but also pivotal in driving the development of safer and more effective therapeutics in the pharmaceutical industry.

Professor Ian S. Gilmore (National Physical Laboratory)

Towards Molecular Volume Imaging with Sub-cellular Resolution

 National Physical Laboratory, Teddington, Middlesex, TW11 0LW, UK

Ian S. Gilmore

Ian.gilmore@npl.co.uk

Volume imaging techniques such as cryogenic focused ion beam electron microscopy (cryo-FIB-SEM) reveal exquisite 3D biological structure of biological material [1,2]. However, there is no molecular information, which would provide powerful complementary insight. In 2017, we introduced the OrbiSIMS technology [3] bringing the performance of an Orbitrap mass spectrometer together with the high-resolution imaging capability of secondary ion mass spectrometry (SIMS). [4] We were able to map the distribution of lipids and neurotransmitters in mouse brain with sub-cellular resolution. We also demonstrated the ability for single-cell metabolic profiling of macrophage cells incubated with the anti-arrhythmia drug, amiodarone, revealing strong heterogeneity of drug uptake. However, with single-cell resolution a clear correlation of drug uptake with upregulation of specific lipids was found. The unique ability of OrbiSIMS to simultaneously provide high-confidence in a molecule’s location and identity has led to strong uptake in a wide range of science and technologies, with now many instruments installed worldwide.

Inspired by advances in Cryo-EM, we introduced the cryo-OrbiSIMS which allows imaging in the native biological state [5]. For example, we demonstrated the ability to undertake in-situ metabolomics of biofilm formation and in collaboration with the Francis Crick Institute we mapped the distribution of metabolites on the cuticular surface of drosophila. [6]

We are now combining cryo-OrbiSIMS with an in situ cryo-FIB to develop protocols for molecular volume imaging. Initial results will be presented as well as novel routes to improve Orbitrap sensitivity by an order of magnitude using a quantum detector. [7]

References

[1]          Schlegel, P et al. Whole-brain annotation and multi-connectome cell typing of Drosophila. Nature 634, 139–152 (2024). Doi: 10.1038/s41586-024-07686-5
[2]          Collinson, L.M. et al. Volume EM: a quiet revolution takes shape. Nat Methods 20, 777–782 (2023) Doi: 10.1038/s41592-023-01861-8
[3]          M K Passarelli et al., The 3D OrbiSIMS-label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power, Nature Methods, 2017. 14 (12): p. 1175
[4]          N. P. Lockyer et al. Secondary ion mass spectrometry, Nat Rev Methods Primers 2024, 4, 32
[5]          J. Zhang et al., Cryo-OrbiSIMS for 3D molecular imaging of a bacterial biofilm in its native state”, Anal. Chem. 2020, 92, 13, 9008–9015.
[6]          C. L. Newell et al, Cryogenic OrbiSIMS Localizes Semi-Volatile Molecules in Biological Tissues, Angewandte Chemie Int. 2020, 59 (41), 18194-18200
[7]          PCT/GB2024/050690 - Improved Spectrometer or Imaging Assembly (2024).

Flash Poster Presentations:

Note: Questions will not be taken during the Flash Presentation sessions; these will be taken during presentation of the accompanying poster. 

1             Ahmed M. A. Abdelhamed
2             Melanie Bailey
3             Ross Chawner
4             Jonathan Ditcham
5             Clare Eynon
6             Markella Loi
7             Chris Mitchell
8             Leonie Mueller
9            Grace Needham
10          Jake Penny
11          Seren Piper
12          Lukas Schlatt
13          Mark Towers
14          Manuel Montano