Biology subjects

Ingargiola, A.

Publications and source records attributed to Ingargiola, A..

5 recordsLinked to original sources

bioRxiv2018

Monte-Carlo Diffusion-Enhanced Photon Inference: Distance Distributions and Conformational Dynamics in single-molecule FRET

Single-molecule Forster Resonance Energy Transfer (smFRET) is utilized to study the structure and dynamics of many bio-molecules, such as proteins, DNA and their various complexes. The structural assessment is based on the well-known Forster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D-A distance distribution and, more recently, interconversion between states in order to extract accurate distance information. It is known that rapid D-A distance fluctuations on the order of the D lifetime (or shorter) can increase the measured mean FRET efficiency and thus decrease the estimated D-A distance. Nonetheless, this effect has been so far neglected in smFRET experiments, potentially leading to biases in estimated distances.\n\nHere we introduce a PDA approach dubbed Monte-Carlo-diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolor detected photons of smFRET experiments taking into account dynamics of D-A distance fluctuations, multiple interconverting states and photo-blinking. Using this approach, we show how different underlying conditions may yield identical FRET histograms and how the additional information from fluorescence decays helps distinguishing between the different conditions. We also introduce a machine learning fitting approach for retrieving the D-A distance distribution, decoupled from the above-mentioned effects. We show that distance interpretation of smFRET experiments of even the simplest dsDNA is nontrivial and requires decoupling the effects of rapid D-A distance fluctuations on FRET in order to avoid systematic biases in the estimation of the D-A distance distribution.

biophysics↗

bioRxiv2017

Optical crosstalk in SPAD arrays for high-throughput single-molecule fluorescence spectroscopy

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability [≤] 1.1 10-3) and is undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

biophysics↗

bioRxiv2017

48-spot single-molecule FRET setup with periodic acceptor excitation

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time.\n\nHere we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

biophysics↗

bioRxiv2016

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.\n\nFinally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

biophysics↗

bioRxiv2016

Applying Corrections in Single-Molecule FRET

Single-molecule Forster resonance energy transfer (smFRET) experiments can detect the distance between a donor and an acceptor fluorophore on the 3-10nm scale. In ratiometric smFRET experiments, the FRET efficiency is estimated from the ratio of acceptor and total signal (donor + acceptor). An excitation scheme involving two alternating lasers (ALEX) is often employed to discriminate between singly- and doubly-labeled populations thanks to a second ratiometric parameter, the stoichiometry S. Accurate FRET and S estimations requires applying three well-known correction factors: donor emission leakage into the acceptor channel, acceptor direct excitation by the donor excitation laser and the \"gamma factor\" (i.e. correction for the imbalance between donor and acceptor signals due to different fluorophores quantum yields and photon detection effciencies).\n\nExpressions to directly correct both raw FRET and S values have been reported in [1] in the context of freely-diffusing smFRET. Here we extend Lee et al. work providing several expressions for the direct excitation coeffcient and highlighting a clear interpretation in terms of physical parameters and experimental quantities. Moreover, we derive a more complete set of analytic expressions for correcting FRET and S. We aim to provide a clear and concise reference for different definitions of correction coeffcients and correction formulas valid for any smFRET experiment both in immobilized and freely-diffusing form.

biophysics↗