Uncovering Hidden Dynamics in Live-Cell Single Molecule Data with Bayesian Statistics

Abstract

Fluorescence microspectroscopy (FMS) with environment-sensitive probes provides information about local molecular surroundings at microscopic spatial resolution. Until recently, only probes exhibiting large spectral shifts due to local changes have been used. Herein, we show that appropriate measuring procedure and data analysis enable nanometer spectral peak position resolution, even for photosensitive fluorophores [1]. The reach of our approach is demonstrated in several examples. The first application shows how we can distinguish lipid vesicles in different lipid phases with two commonly used polarity-sensitive probes. A synthesized NBD-based fatty acid red-shifted its emission maximum by 1.5-2 nm going from gel to liquiddisordered phase in DPPC. Between these two phases Laurdan exhibits a large 50 nm red-shift. We therefore chose a more challenging combination -gel and liquid ordered phase, realized by DPPC and DPPC/Chol (40 mol%), respectively, where we were able to detect a 3 nm blue-shift with Laurdan [1]. The second example shows application of a synthesized rhodamine-based pH-activatable probe that is sensitive to aggregation. We studied a receptor-mediated internalization in dendritic cells and measured a 3 nm aggregation-induced emission spectral shift due to probe accumulation in endosomes and lysosomes [2,3]. The results show that peak position resolution, characteristic for spectrofluorimetric measurements on bulk samples, could readily be achieved at micrometer spatial scale.[1] I. Urban ci c, Z.