Theoretical Comparison of Real-Time Single Particle Tracking Techniques

Abstract

Super-resolution microscopy and single molecule fluorescence spectroscopy require complementary and mutually exclusive experimental strategies that optimize either spatial or temporal resolution, making their combined implementation a challenge. With a GPU-driven, camera-based measurement strategy we demonstrate that it is possible to achieve this in 5 minutes (including measurement time) using the epidermal growth factor receptor (EGFR) as our model. EGFR, a transmembrane receptor tyrosine kinase, is a crucial regulator of proliferation and survival, and its mutations and dysregulated signalling have been implicated in many cancers. However, a clear understanding of its oligomerization state, dynamics, and interactions with the actin cytoskeleton and other plasma membrane components has been elusive, and we have investigated these in live CHO-K1 cells using TIRF microscopy. Simultaneous dual-channel spatiotemporal super-resolution measurements were performed using the actin marker Lifeact along with EGFR. By optimizing the extraction of various parameters through spatial or temporal binning, a single data set is used for multi-parametric analysis involving imaging fluorescence correlation spectroscopy (ImFCS) to study EGFR dynamics, number and brightness analysis (N&B) to determine EGFR oligomerization, FCS diffusion law to determine sub-resolution EGFR localization, and super-resolution radial fluctuations (SRRF) to super-resolve actin fibres. Cross-correlation analysis between any two parameters provides new biological knowledge that cannot be obtained from separate sequential measurements. A combination of measurements involving resting cells, ligand addition, drug treatments, and EGFR mutants revealed that the domain localization of EGFR in membranes is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions, but is largely independent of EGFR-actin interactions. Our approach does not require specialized instrumentation, can be implemented with other combinations of fluorescence techniques, and thus is easily applicable and useful to answer a wide variety of biological questions.