Track to the Future: Improving Selection of Intensity Tracks for FLImP Super-Resolution Imaging

Description

Lung cancer is a major cause of cancer-related deaths globally, with Non-Small Cell Lung Cancer (NSCLC) representing most cases. Mutations in Epidermal Growth Factor Receptors (EGFRs) contribute to NSCLC progression by influencing protein clustering on the cell membrane. Analysing EGFR spatial distribution provides insights into cancer development and drug resistance, which could help improve therapeutic strategies.

Fluorescence Localisation Imaging with Photobleaching (FLImP; Needham et al., 2013) is a super-resolution technique capable of resolving fluorescently labelled EGFR molecules beyond the diffraction limit. FLImP produces staircase-like intensity tracks as fluorophores transitions through fluorescence states, which can be analysed using MCMC changepoint estimation to determine active fluorophore numbers per frame. From this information, EGFR positions can be estimated from two-dimensional images. A single FLImP series can generate up to 10,000 tracks, though only a small fraction meet the quality requirements for computationally intensive downstream analysis (Iyer et al., 2024). However, current track selection methods rely on inefficient, heuristic filtering, using less than 1% of collected data.

We introduce a refined methodology for post-processing track selection, incorporating uncertainty estimates from MCMC analysis and exploring hidden Markov models for fluorophore state identification, with progress in individual fluorophore labelling discussed. The methodology is validated using simulated data and compared with the heuristic gold standard (Iyer et al., 2024), to evaluate its performance. Enhancing the efficiency of this approach while maintaining explainability, allows faster acquisition of EGFR molecular fingerprints using fewer cells and paves the way for two-dimensional molecular reconstruction; a step towards the clinical application of FLImP.

Needham, Sarah R., et al. "Measuring EGFR Separations on Cells with ∼10nm Resolution via Fluorophore Localization Imaging with Photobleaching." PloS One 8.5 (2013): e62331.

Iyer, R. Sumanth, et al. “Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.” Nature Communications 15 (2024): 2130

Period	02 Sept 2025
Event title	RSS International Conference 2025
Event type	Conference
Location	United KingdomShow on map
Degree of Recognition	International