Abstract
Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.
Highlights
Methods to capture static molecular structures, such as superresolution microscopy [1,2,3], provide only snapshots of life in time
Our goal is to characterize quantities that describe molecular dynamics, especially dynamics encountered in biological samples, such as diffusion coefficients, at the data-acquisition timescales of conventional single-focus confocal setups
A single photon arriving at a detector mounted to a confocal microscope encodes information that reports on the fastest timescale achievable for spectroscopic and imaging applications [11,80]
Summary
Methods to capture static molecular structures, such as superresolution microscopy [1,2,3], provide only snapshots of life in time. Life is dynamical and obtaining a picture of life in action—one that captures diffraction-limited biomolecules as they move, assemble into, and disassemble from larger bimolecular complexes—remains an important challenge [4]. The creative insights directly leading to fluorescence correlation spectroscopy (FCS) [5,6]—and. Related methods such as FCS FRET [7,8] and FCCS [9]— have shown that deciphering dynamical information from molecules, often biomolecules, does not demand spatial resolution or spatial localization. The key is to inhomogeneously illuminate a sample over a small volume
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