Experimental and computational methods for two-colour single-particle tracking: a toolset for unveiling single biomolecules interactions

Single-particle tracking (SPT) is a powerful technique to investigate biological questions at the single-molecule level, providing a full characterization of the spatio-temporal organization of living systems in physiological contexts. One-colour applications have improved in recent years thanks to advances in several aspects, from labelling strategies to tracking algorithms. Multicolour extensions of the technique can provide quantitative descriptions of biological interactions, but they are still limited.I found shortcomings in the literature related to three main aspects: limitations in the signal-to-noise ratio in total internal reflection (TIRF) microscopy, especially when observing fast-diffusing receptors; lack of a robust method to determine fluorescence labelling efficiency; lack of tracking algorithms specific for multicolor applications. These shortcomings challenge two-colour SPT studies, as I discuss in Chapter 1 of the thesis, limiting the fields and conditions of applications or the accuracy of the results. I worked on all these points, developing methods able to overcome current limitations and make SPT feasible in more demanding conditions.Chapter 2 describes our strategies for implementing a sensitive multi-channel TIRF microscopy system. We show that several sources of undesirable background still exist in a typical TIRF setup; in particular, standard optical glasses used for microscopy cover glasses emit autofluorescence proved significant for the experiments of our interest. We identify an alternative optical material and describe its use for the first time as an alternative cover glass for TIRF, achieving significant SNR improvements in a multichannel configuration. Our strategies are applied to visualize fast p75NTR receptors labeled by two fluorophores on the membrane of living cells, achieving reliable, simultaneous two-color SPT.Chapter 3 presents the method we developed to quantify fluorescent-labeling efficiency. It operates in the same conditions as the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We applied the method to the labeling of the membrane-receptor TrkA through 4'-phosphopantetheinyl transferase Sfp in living cells, finding, for the first time for this kind of labeling, the conditions for demanding single-molecule studies at high degrees of labeling and low aspecific binding.Chapter 4 focuses on some computational aspects. We describe our simulation algorithms for SPT and our algorithm for two-colour tracking; the latter exploits parallel programming to integrate the information available in two different channels. We show promising results for obtaining more accurate tracking results compared to previous approaches found in the literature.