Fluorescence Axial Localization with Nanometer Accuracy and Precision

Li, Hui; Yen, Chi-Fu; Sivasankar, Sanjeevi

doi:10.1021/nl301542c

Cited by 14 publications

(13 citation statements)

References 27 publications

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“… 59 , 60 FRET detection on the basis of donor lifetime is more effective and less sensitive to local environment fluctuations, 26 especially in AFM tip-enhanced single-molecule spectroscopic and imaging measurements where amplified fluorescence signal by metal tip reflection exists. 25 , 61 In the photon stamping spectroscopy, each detected photon is stamped with two parameters: a chronic arrival time and a delay time related to femtosecond laser pulse excitation. In this work, we treat photons distributions detected in each 10 ms (ms) bins as a Poisson distribution which gives the mean of delay times in each distribution as the lifetime τ DA .…”

Section: Methodsmentioning

confidence: 99%

Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

2014

J. Phys. Chem. B

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Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental understanding of the enzymatic reaction turnover dynamics associated with overall enzyme as well as the specific active-site conformational fluctuations that are not identifiable and resolvable in the conventional ensemble-averaged experiment.

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Section: Methodsmentioning

confidence: 99%

Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

2014

J. Phys. Chem. B

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“…The accuracy and precision of our method are within the size of individual proteins (∼5 nm). Nanometre accuracy and precision have been reported (Li et al ., ) before with AFM under specific experimental configurations; in contrast, the accuracy and precision of our method is comparable at ambient and easy to use configurations facilitating high resolution tracking with commonly used TIRF microscopes. By taking advantage of multiple fiducial markers, this method is robust against tracking errors such as missing points, image processing artefacts, interference from diffusing labels and non‐Gaussian noise sources.…”

Section: Resultsmentioning

confidence: 97%

Estimation of microscope drift using fluorescent nanodiamonds as fiducial markers

Colomb

Czerski

et al. 2017

Journal of Microscopy

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Fiducial markers are used to correct the microscope drift and should be photostable, be usable at multiple wavelengths and be compatible for multimodal imaging. Fiducial markers such as beads, gold nanoparticles, microfabricated patterns and organic fluorophores lack one or more of these criteria. Moreover, the localization accuracy and drift correction can be degraded by other fluorophores, instrument noise and artefacts due to image processing and tracking algorithms. Estimating mechanical drift by assuming Gaussian distributed noise is not suitable under these circumstances. Here we present a method that uses fluorescent nanodiamonds as fiducial markers and uses an improved maximum likelihood algorithm to estimate the drift with both accuracy and precision within the range 1.55-5.75 nm.

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“…Solutions are made by suspending Cy3 dye in glycerolbuffer (pH 7.5, 10 mM Tris-HCl, 100 mM NaCl, and 10 mM KCl, 2.5 mM CaCl 2 ) at various v=v, to a final concentration of either 100 pM or 1 nM. The solution is placed in a glass-bottomed fluid cell, assembled on a custom-designed confocal microscope [73] and a 532-nm laser beam is focused to a diffraction-limited spot on the glass coverslip of the fluid cell using a 60×, 1.42 N.A., oilimmersion objective (Olympus). In our setup, the laser beam is focused at the glass-water-glycerol interface and the beam is refocused by visual inspection at the beginning of every measurement.…”

Section: Acquisition Of Experiments Data For Figs 8-12mentioning

confidence: 99%

Pitching Single-Focus Confocal Data Analysis One Photon at a Time with Bayesian Nonparametrics

et al. 2020

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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.

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Fluorescence Axial Localization with Nanometer Accuracy and Precision

Cited by 14 publications

References 27 publications

Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

Estimation of microscope drift using fluorescent nanodiamonds as fiducial markers

Pitching Single-Focus Confocal Data Analysis One Photon at a Time with Bayesian Nonparametrics

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