Burst Integrated Fluorescence Lifetime Research Articles

A Multiscalar Framework describes Fluorescence and FRET of Fluctuating Molecular Species and Resolves Kinetic Networks

A combination of multi parameter fluorescence detection (MFD) with structure based fluorescence models is presented, to capture and describe fluorescence and FRET between a donor (D) and acceptor (A) dye of fluctuating macromolecules over more than five orders of magnitude from picoseconds to seconds with Angstrom resolution. The presented top down approach combines molecular models with established fluorescence techniques such as time correlated single photon counting, burst integrated fluorescence lifetime analysis, filtered fluorescence correlation spectroscopy (fFCS), and photon distribution analysis in a joined framework and thus facilitates the analysis and interpretation of fluorescence experiments. Fluorescence and FRET on the picoseconds to nanosecond regime is described by combining atomistic models with a coarse grained representation of the dyes. Their conformational space is quantified by coarse grained accessible volume simulations while Brownian dynamics simulations capture transient effects of FRET and fluorescence quenching. Assuming dynamic quenching and FRET are decoupled, the first two modes of the DA-distance distribution are determined for single molecules to conveniently reveal macromolecular kinetics on the milli- and sub-millisecond kinetics by MFD histograms. Structural models are projected to yield parametric equations of single-molecule observables. This serves as a visual guide to analyse MFD- histograms. Numeric integration of the chemical master equation analyses MFD-histograms and quantifies in combination with fFCS kinetic networks of dynamically exchanging macromolecular conformations in the sub-microsecond to millisecond regime. A joint analysis of multiple fluorescence decays by structure based patterns resolved chemical equilibria in live cell. In future, such holistic approaches may exploit the information contained in the fluorescence signal and connect dynamic molecular structural models with kinetic and equilibrium networks of biomolecules to picture molecular machines in living cells.

Real-time fluorescence lifetime actuation for cell sorting using a CMOS SPAD silicon photomultiplier.

Time-correlated single photon counting (TCSPC) is a fundamental fluorescence lifetime measurement technique offering high signal to noise ratio (SNR). However, its requirement for complex software algorithms for histogram processing restricts throughput in flow cytometers and prevents on-the-fly sorting of cells. We present a single-point digital silicon photomultiplier (SiPM) detector accomplishing real-time fluorescence lifetime-activated actuation targeting cell sorting applications in flow cytometry. The sensor also achieves burst-integrated fluorescence lifetime (BIFL) detection by TCSPC. The SiPM is a single-chip complementary metal-oxide-semiconductor (CMOS) sensor employing a 32×32 single-photon avalanche diode (SPAD) array and eight pairs of time-interleaved time to digital converters (TI-TDCs) with a 50 ps minimum timing resolution. The sensor's pile-up resistant embedded center of mass method (CMM) processor accomplishes low-latency measurement and thresholding of fluorescence lifetime. A digital control signal is generated with a 16.6 μs latency for cell sorter actuation allowing a maximum cell throughput of 60,000 cells per second and an error rate of 0.6%.

Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening.

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry

The other side of the coin: time-domain fluorescence lifetime in flow.

Keywords: fluorescence lifetime; flow cytometry; time-correlated single photon counting (TCSPC); burst integrated fluorescence lifetime (BIFL); time-tagged time-resolved (TTTR); fluorescence resonance energy transfer (FRET)

Heterogeneity in binary mixtures of dimethyl sulfoxide and glycerol: Fluorescence correlation spectroscopy

Diffusion of four coumarin dyes in a binary mixture of dimethyl sulfoxide (DMSO) and glycerol is studied using fluorescence correlation spectroscopy (FCS). The coumarin dyes are C151, C152, C480, and C481. In pure DMSO, all the four dyes exhibit a very narrow (almost uni-modal) distribution of diffusion coefficient (Dt). In contrast, in the binary mixtures all of them display a bimodal distribution of Dt with broadly two components. One of the components of D(t) corresponds to the bulk viscosity. The other one is similar to that in pure DMSO. This clearly indicates the presence of two distinctly different nano-domains inside the binary mixture. In the first, the micro-environment of the solute consists of both DMSO and glycerol approximately at the bulk composition. The other corresponds to a situation where the first layer of the solute consists of DMSO only. The burst integrated fluorescence lifetime (BIFL) analysis also indicates presence of two micro-environments one of which resembles DMSO. The relative contribution of the DMSO-like environment obtained from the BIFL analysis is much larger than that obtained from FCS measurements. It is proposed that BIFL corresponds to an instantaneous environment in a small region (a few nm) around the probe. FCS, on the contrary, describes the long time trajectory of the probes in a region of dimension ~200 nm. The results are explained in terms of the theory of binary mixtures and recent simulations of binary mixtures containing DMSO.

Role of Ionic Liquid on the Conformational Dynamics in the Native, Molten Globule, and Unfolded States of Cytochrome C: A Fluorescence Correlation Spectroscopy Study

The role of a room temperature ionic liquid (RTIL, [pmim][Br]) on the size and conformational dynamics of a protein, horse heart cytochrome c (Cyt C) in its native, molten globule (MG-I and II), and unfolded states is studied using fluorescence correlation spectroscopy (FCS). For this purpose, the protein was covalently labeled by a fluorescent dye, Alexa Fluor 488. It is observed that the addition of the RTIL leads to an increase in the hydrodynamic radius (r(H)) of the protein, Cyt C in the native or MG-I state. In contrast, the addition of RTIL causes a decrease in the size (hydrodynamic radius, r(H)) of Cyt C unfolded by GdnHCl or MG-II state. The decrease in size indicates the formation of a relatively compact structure. We detected two types of conformational relaxation of the protein. The shorter relaxation time component (~3-5.5 μs) corresponds to the protein folding or intrachain contact formation, while the relatively longer time component (~63-122 μs) may be assigned to the motion of the protein side chains or concerted chain dynamics. The burst integrated fluorescence lifetime histograms indicate that the increase in size of the protein is accompanied by an increase in the contribution of the shorter component (~0.3-0.4 ns) with a concomitant decrease of the contribution of the longer component (~2.8-3.6 ns). An opposite trend is observed during the decrease in size of the protein.

Study of γ-Cyclodextrin Host–Guest Complex and Nanotube Aggregate by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) has been used to study the formation of large nanotube aggregates involving γ-cyclodextrin (γ-CD) and coumarin 153 (C153). It is observed that the length of a γ-CD:C153 nanotube aggregate is ∼770 nm. This is ∼480 times larger than the length of a 1:1 γ-CD:C480 complex (∼1.6 nm) and ∼950 times that of a γ-CD. This implies that 950 γ-CD units are noncovalently attached in the γ-CD:C153 aggregate. Binding constants (K(b)) of both the dyes to γ-CD were obtained from the fluctuation in fluorescence intensity. The rate of association and dissociation are obtained from the inverse of τ(off) and τ(on), respectively. The binding constant for the 1:1 γ-CD:C480 complex is ∼1000 M(-1). The burst integrated fluorescence lifetime (BIFL) histogram reveals presence of three distinct lifetime 1.8 ns (18%), 2.8 ns (69%), 3.2 ns (13%). These three lifetimes correspond to C153 present in bulk water and at the end and middle of the γ-CD:C153 nanotube aggregate, respectively. The lifetime of C480 in the 1:1 γ-CD:C480 complex is found to be 3.7 ns.

A Bayesian method for single molecule, fluorescence burst analysis

There is currently great interest in determining physical parameters, e.g. fluorescence lifetime, of individual molecules that inform on environmental conditions, whilst avoiding the artefacts of ensemble averaging. Protein interactions, molecular dynamics and sub-species can all be studied. In a burst integrated fluorescence lifetime (BIFL) experiment, identification of fluorescent bursts from single molecules above background detection is a problem. This paper presents a Bayesian method for burst identification based on model selection and demonstrates the detection of bursts consisting of 10% signal amplitude. The method also estimates the fluorescence lifetime (and its error) from the burst data.

Identification of Single Molecules in Aqueous Solution by Time-Resolved Fluorescence Anisotropy

Using a confocal epi-illuminated microscope with a polarizing beam splitter and dual-channel detection of single-molecule fluorescence induced by pulsed laser excitation, a new application of the three-dimensional, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) is introduced. BIFL allows simultaneous registration of fluorescence intensity, lifetime, and anisotropy. It is shown to be well-suited to identify the freely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fluorescent Protein via their characteristic fluorescence anisotropy using a time-resolved analysis. Furthermore, data analysis is discussed and rotational correlation times of single molecules are determined. Applications for multidimensional single-molecule identification are outlined.

Quantitative Identification of Different Single Molecules by Selective Time-Resolved Confocal Fluorescence Spectroscopy

Using a confocal epi-illuminated microscope together with a pulsed laser, new applications of the recently developed, real-time spectroscopic technique BIFL (burst integrated fluorescence lifetime) are introduced. BIFL registers two different types of information on every detected photon with regard to the macroscopic time scale of a measurement and to the fluorescence lifetime. Thus, it is shown to be well suited to identify freely diffusing single dye molecules via their characteristic fluorescence lifetime. This allows for selective counting of dye molecules in an open volume element and opens up the possibility to quantify the relative concentration of the dye molecules, using a recently derived theoretical model, which analyzes the obtained burst size distribution of a sample survey. A closed theory is presented to calculate the probability of a specific dye to cause a fluorescence burst containing a certain number of detected photons. It considers the distribution of the excitation irradiance over t...

Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy.

A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (approximately 10 fl) of a confocal epi-illuminated set-up. In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules are registered and further subjected to selective burst analysis. The two-dimensional BIFL data allow the identification and detection of different temporally resolved conformational states. A complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity. The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.