Fluorescence Lifetime Distribution Research Articles

Fluorescence imaging of nanobiomaterials: Thioflavin T in electrospun poly(vinyl alcohol) fibres

Nanoindentation has been widely used to characterise the mechanical properties of various materials. As an alternative, we recently explored a new technique with which the Young's modulus of soft materials can be obtained using environment-sensitive fluorescent dyes. The fluorescence technique has an advantage over nanoindentation in that it can non-destructively measure the physical property of specific positions of nanomaterials. In this work, we have employed fluorescence lifetime imaging microscopy (FLIM) that utilises time-correlated single photon counting and confocal microscopy to investigate the rigidity image of nanostructured materials. ThT-embedded poly(vinyl alcohol) (PVA) polymer films and nanofibres were prepared by spin coating and electrospinning, respectively, and the fluorescence lifetime distribution was obtained by FLIM. It appears that the Thioflavin T (ThT) dye, which was originally used to detect amyloid fibrils, is an excellent probe for our purpose because the excited dynamics of ThT depends on the mechanical properties of surrounding matrices. That is, the time scale of the internal twisting motion of electronically excited ThT was well correlated to the mechanical properties of polymer nanostructures. Our approach opens a new way with which the mechanical properties of advanced nanobiomaterials can be obtained using the modulus-probing dye.

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.

Investigating protein-protein interactions in the plant endomembrane system using multiphoton-induced FRET-FLIM.

Real-time noninvasive fluorescence-based protein assays enable a direct access to study interactions in their natural environment and hence overcome the limitations of other methods that rely on invasive cell disruption techniques. The determination of Förster resonance energy transfer (FRET) by means of fluorescence lifetime imaging microscopy (FLIM) is currently the most advanced method to observe protein-protein interactions at nanometer resolution inside single living cells and in real-time. In the FRET-FLIM approach, the information gained using steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction. The combination of confocal laser scanning microscopy with the sensitive advanced technique of time-correlated single photon counting allows the mapping of the spatial distribution of fluorescence lifetimes inside living cells on a pixel-by-pixel basis that is the same as the fluorescence image. Moreover, the use of multiphoton excitation particularly for plant cells provides further advantages such as reduced phototoxicity and photobleaching. In this protocol, we briefly describe the instrumentation and experimental design to study protein interactions within the plant endomembrane system, with a focus on the imaging of plant cells expressing fluorescent proteins and acquisition and analysis of fluorescence lifetime resolved data.

Critical Evaluation of the Two-State Model Describing the Equilibrium Unfolding of the PI3K SH3 Domain by Time-Resolved Fluorescence Resonance Energy Transfer

It appears that equilibrium unfolding transitions of many small proteins can be described as two-state transitions, because the probes commonly used to measure such transitions cannot detect the underlying heterogeneity inherent in protein folding and unfolding reactions. Time-resolved fluorescence or Forster resonance energy transfer (TRFRET) measurements have the potential to uncover such heterogeneity and to test the cooperativity of protein folding reactions. Here, TRFRET measurements have been used to study the equilibrium unfolding of the SH3 domain of PI3 kinase. The single tryptophan residue (W53) was used as the FRET donor, and a covalently attached thionitrobenzoate moiety at either of two sites (C17 and C70) was used as the FRET acceptor. The individual lifetime and amplitude components estimated from fitting the fluorescence decay kinetics to the sum of three or four exponentials were determined over a range of denaturant concentrations. The equilibrium unfolding transitions reported by these components were found to be noncoincident, suggesting the presence of multiple conformations in equilibrium during the course of unfolding. Fluorescence lifetime distributions were also generated by the model-free maximum entropy method of analysis. Different segments of the protein were found to show differences in the expansion of the native state at low denaturant concentrations, suggestive of gradual structural transitions. The unfolded protein was found to swell at increasingly high denaturant concentrations. The evolution of the fluorescence lifetime distributions with increasing denaturant concentration was also found to be incompatible with a two-state equilibrium unfolding model.

Fluorescence lifetime distributions report on protein destabilisation in quenching experiments

Tryptophan is the most often investigated intrinsic fluorophore due to its abundance in proteins and its sensitivity to different environmental conditions. Fluorescence quenching is a powerful method to study proteins and acrylamide is a frequently applied quencher in these investigations. Quenching experiments are sometimes distorted by the undesired protein–quencher interactions that can result in a misinterpretation of the results. Here we focused on the identification of the possible side-effects of acrylamide applying fluorescence lifetime measurements. To provide reference data for protein denaturation the fluorescence parameters were also recorded in the presence of different concentrations of guanidine hydrochloride. In circular dichroism experiments we characterized directly the acrylamide effect on the tertiary structure of the proteins. According to the obtained data in experiments with seven tryptophan-containing proteins the full width at half maximum (FWHM) of the fluorescence lifetime distribution is an appropriate parameter to monitor the undesired effects of acrylamide on the proteins.

Phosphorylated Phospholamban Stabilizes a Distinct Compact Conformation of Sarco(endo)Plasmic Reticulum Ca-ATPase

Sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is inhibited by the 52 amino acid protein phospholamban (PLB). Inhibition of SERCA is relieved when PLB is phosphorylated by protein kinase A (PKA). To better understand the mechanism of this regulatory mechanism on SERCA structure, we designed a “2-color” SERCA labeled with donor and acceptor fluorescent proteins in the A- and N-domain. Intramolecular fluorescence resonance energy transfer (FRET) between these two domains was quantified as an index of SERCA conformation. Fluorescence lifetime distribution analysis (FLDA) showed that SERCA sampled three discrete conformations in the absence of PLB or in the presence of non-phosphorylatable PLB (S16A). Co-transfection of SERCA with phosphomimetic PLB (S16E) revealed an additional state characterized by a highly compact conformation. This unique state was only observed under conditions of high intracellular calcium. The data support the hypothesis that PLB remains bound to SERCA after phosphorylation by PKA, inducing the pump to assume a very compact E1 conformation.

Structure Transitions of the Sodium, Potassium-ATPase Investigated by Intramolecular FRET

The sodium, potassium-ATPase (NKA) is an important ion pump responsible for the generation of electrochemical gradients across eukaryotic plasma membranes. This protein is a high-value therapeutic target for the treatment of heart failure and several carcinomas. We fused fluorescent proteins to the N and A domains of NKA and investigated conformational changes by quantifying intramolecular FRET. Fluorescence lifetime distribution analysis (FLDA) revealed a decrease in FRET with the addition of 25 mM KCl or 1mM ouabain, suggesting a structure transition to a more open conformation. In addition, the low-FRET conformations show a wider lifetime distribution than high-FRET architectures, which suggests that the open structure has a more dynamic cytoplasmic headpiece than the closed conformation. Ongoing experiments will investigate the voltage dependence of FRET changes and NKA structural heterogeneity.

SERCA Dynamic Structures Characterized with FLDA-FRET Method

The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) undergoes conformational changes between discrete enzymatic substates during its catalytic cycle. To investigate the dynamic structures of SERCA, we fused donor and acceptor fluorescent proteins to the A and N domains of SERCA and quantified intramolecular FRET. We measured the distribution of FRET states using fluorescence lifetime distribution analysis (FLDA). This approach was validated with standard samples including dyes and Cerulean-Venus fusion controls. We found SERCA has a high degree of cytoplasmic domain structural disorder when it assumes open structures, manifest as a low FRET efficiency and a wide FRET distribution. In contrast, compact conformations were characterized by a well-defined, rigid architecture, yielding high FRET efficiency, with a narrow FRET distribution. The pump inhibitor thapsigargin abolished closed structures and stabilized SERCA in open conformations, but did not decrease the disorder of SERCA cytoplasmic domains. FRET was increased and disorder was decreased by low temperature. It is possible that high-FRET, tightly closed conformations are stabilized by domain-domain contacts, while open conformations are unconstrained and floppy. The functional consequence of a dynamic open architecture may be enhanced mobility of the nucleotide binding domain during its Brownian search for ATP substrate.

Depth-Dependent Heterogeneity in Membranes by Fluorescence Lifetime Distribution Analysis.

Biological membranes display considerable anisotropy due to differences in composition, physical characteristics, and packing of membrane components. In this Letter, we have demonstrated the environmental heterogeneity along the bilayer normal in a depth-dependent manner using a number of anthroyloxy fatty acid probes. We employed fluorescence lifetime distribution analysis utilizing the maximum entropy method (MEM) to assess heterogeneity. Our results show that the fluorescence lifetime heterogeneity varies considerably depending on fluorophore location along the membrane normal (depth), and it is the result of the anisotropic environmental heterogeneity along the bilayer normal. Environmental heterogeneity is reduced as the reporter group is moved from the membrane interface to a deeper hydrocarbon region. To the best of our knowledge, our results constitute the first experimental demonstration of anisotropic heterogeneity in bilayers. We conclude that such graded environmental heterogeneity represents an intrinsic characteristics of the membrane bilayer and envisage that it has a role in the conformation and orientation of membrane proteins and their function.

Monitoring the distribution of warfarin in blood plasma.

Warfarin is an anticoagulant drug extensively used in the treatment and prevention of thrombotic disorders. Previous studies have shown that warfarin binds extensively to blood plasma proteins and that only a small fraction of the drug is unbound and thus available for therapeutic function. Both warfarin's narrow therapeutic window and the susceptibility of anticoagulant function to patient-dependent factors necessitate regular monitoring. In this study, we have shown that the lifetimes for each of the various bound and free forms of the drug in blood plasma can be quantified in situ by time-correlated single-photon counting fluorescence spectroscopy over the clinically significant concentration range. A relationship between the blood coagulation and the distribution of fluorescence lifetimes was observed. The in situ detection of clinically relevant concentrations of warfarin in its respective bound and unbound forms could provide a prognostic tool for use in patient treatment.

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET

In single-molecule FRET experiments with pulsed lasers, not only the colors of the photons but also the fluorescence lifetimes can be monitored. Although these quantities appear to be random, they are modulated by conformational dynamics. In order to extract information about such dynamics, we develop the theory of the joint distribution of FRET efficiencies and fluorescence lifetimes determined from bins (or bursts) of photons. Our starting point is a rigorous formal expression for the distribution of the numbers of donor and acceptor photons and donor lifetimes in a bin that treats the influence of conformational dynamics on all timescales. This formula leads to an analytic result for a two-state system interconverting on a timescale slower than the interphoton time and to an efficient simulation algorithm for multistate dynamics. The shape of the joint distribution contains more information about conformational dynamics than the FRET efficiency histogram alone. In favorable cases, the connectivity of the underlying conformational states can be determined directly by simple inspection of the projection of the joint distribution on the efficiency-lifetime plane.

G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots.

The use of time-resolved fluorescence measurements in studies of telomeric G-quadruplex folding and stability has been hampered by the complexity of fluorescence lifetime distributions in solution. The application of phasor diagrams to the analysis of time-resolved fluorescence measurements, collected from either frequency-domain or time-domain instrumentation, allows for rapid characterization of complex lifetime distributions. Phasor diagrams are model-free graphical representations of transformed time-resolved fluorescence results. Simplification of complex fluorescent decays by phasor diagrams is demonstrated here using a 2-aminopurine substituted telomeric G-quadruplex sequence. The application of phasor diagrams to complex systems is discussed with comparisons to traditional non-linear regression model fitting. Phasor diagrams allow for the folding and stability of the telomeric G-quadruplex to be monitored in the presence of either sodium or potassium. Fluorescence lifetime measurements revealed multiple transitions upon folding of the telomeric G-quadruplex through the addition of potassium. Enzymatic digestion of the telomeric G-quadruplex structure, fluorescence quenching and Förster resonance energy transfer were also monitored through phasor diagrams. This work demonstrates the sensitivity of time-resolved methods for monitoring changes to the telomeric G-quadruplex and outlines the phasor diagram approach for analysis of complex time-resolved results that can be extended to other G-quadruplex and nucleic acid systems.

Investigating the Effect of Redox Agents on Structural Re-Arrangement of the BK Channel RCK1-RCK2 Linker using Fluorescence Lifetime Imaging Microscopy

Alternative splicing generates considerable functional diversity of large conductance calcium- and voltage-activated potassium (BK) channels. For example, inclusion of the STREX (STRess-activated EXon) insert into the unstructured cytosolic linker between the two RCK domains of the channel generates a cysteine rich domain (CRD) that confers intrinsic hypoxia sensitivity to the channel [1].We have utilized YFP-mCFP fluorescent fusion proteins of the CRD and exploited these to examine conformational rearrangements in the CRD in response to hypoxia and redox agents using fluorescence lifetime imaging microscopy (FLIM). The fusion protein, expressed in HEK293 cells, is targeted to the plasma membrane and responds to acute hypoxia (10 minute exposure at <5% oxygen) with a significant shift in fluorescence lifetime distribution. Pre-treatment with the oxidizing agent DTNB (1mM) appeared to cause a shortening in the lifetime distribution that did not block the hypoxia induced shift in fluorescence lifetimes (n=4 for each). Treatment with reducing agents NEM (1mM) or sodium sulfide (1mM) did not lead to significant changes in fluorescent lifetime nor did they block the hypoxia response (n=3-5 for each). The effects of redox manipulation on hypoxia sensitivity were recapitulated in patch clamp assays of channel activity. Together this data supports the hypothesis that the STREX channel hypoxia response can not be mimicked by redox agents and that FLIM, in conjunction with patch clamp electrophysiology, allows the monitoring of conformational rearrangements and investigation of the structure-function relationship of the linker region between the channel RCK domains.[1] McCartney, C.E., McClaffert, H., Huibant, J.M., Rowan, E.G., Shipston, M.J, and Rowe. I.C. (2005) A cysteine-rich motif confers hypoxia sensitivity to mammalian large conductance voltage- and Ca-activated K (BK) channel alpha-subunits. Proc Natl Acad Sci USA 102:17870-17876

Single-molecule fluorescence dynamics of a butadiyne-linked porphyrin dimer: the effect of conformational flexibility in host polymers

We have investigated the single-molecule fluorescence dynamics of a butadiyne-linked porphyrin dimer (Z2B) depending on the density of the poly(methyl methacrylate) (PMMA) matrix (5, 10, 25, and 50 mg ml(-1)). By recording single-molecule fluorescence intensity trajectories and fluorescence lifetimes, we observed more frequent one-step photobleaching behavior, less frequent on-off behavior, and narrower fluorescence lifetime distributions in lower densities of PMMA polymer. In contrast, more enhanced photostability was observed in higher densities of PMMA polymer. These results are explained by a difference in the molecular surroundings depending on the change in PMMA polymer density, suggesting that the individual photophysical properties of Z2B are strongly associated with their conformations and molecular surroundings in the solid state. Our studies will provide further information on the structure/surroundings relationship of single molecules in the solid state.

Spatial Fluorescence Inhomogeneities in Light-Emitting Conjugated Polymer Films

Scanning confocal time-resolved fluorescence imaging measurements have been performed on light-emitting conjugated polymer thin films cast from a range of solvents. We have observed inhomogeneities in the emission spectra and fluorescence decay profiles at various micrometer-sized regions within these films. Some regions are observed to exhibit longer fluorescence decay times and emission at shorter wavelengths than typically reported for such films. These regions are associated with nonaggregated polymer structures. Solvent effects on the photophysical properties of conjugated polymer films were also investigated. The dissimilarity of fluorescence lifetime distributions and emission spectra for films deposited from different solvents shows the diversity of the configurations within films and a dependence/memory of the polymer conformation on the solvent from which the film was cast.

Investigating Structural Re-Arrangement of the BK Channel Rck1-Rck2 Linker Using Fluorescence Lifetime Imaging Microscopy

Alternative splicing generates considerable functional diversity of large conductance calcium- and voltage-activated potassium (BK) channels. For example, inclusion of the STREX (STRess-activated EXon) insert into the unstructured cytosolic linker between the two RCK domains of the channel generates a cysteine rich domain (CRD) that confers intrinsic hypoxia sensitivity to the channel [1]. Here, we have developed YFP-mCFP fluorescent fusion proteins of the CRD and exploit these to examine conformational rearrangements in the CRD in response to hypoxia using fluorescence lifetime imaging microscopy (FLIM). The fusion protein, expressed in HEK293 cells, is targeted to the plasma membrane and responds to acute hypoxia (10 minute exposure at <5% oxygen) with a significant shift in fluorescence lifetime distribution (n=7). Pre-treatment with either oxidizing or reducing agents did not block the hypoxia induced shift in fluorescence lifetimes (n=4). In contrast, site directed mutagenesis of the cysteine residues within STREX, which we had previously shown to confer functional hypoxia sensitivity to the channel, abolished the hypoxia induced shift in lifetimes (n=6). The effects of cysteine mutagenesis and REDOX manipulation on hypoxia sensitivity were recapitulated in patch clamp assays of channel activity supporting a role for hypoxia-induced shifts in CRD conformation with changes in channel function. These findings suggest that FLIM, in conjunction with patch clamp electrophysiology, will be an important approach to monitor conformational rearrangements and investigate the structure-function relationship of the important unstructured linker region between the channel RCK domains. [1] McCartney, C.E., McClaffert, H., Huibant, J.M., Rowan, E.G., Shipston, M.J, and Rowe. I.C. (2005) A cysteine-rich motif confers hypoxia sensitivity to mammalian large conductance voltage- and Ca-activated K (BK) channel alpha-subunits. Proc. Natl. Acad. Sci. USA 102:17870-17876

Conformational Changes of SERCA Revealed by Intramolecular FRET

The first structures of the sarcoendoplasmic reticulum ATPase (SERCA) determined by X-ray crystallography suggested that pump undergoes a large conformational change during catalytic cycling. The transition from the E1 (Ca-bound) state to the E2 (Ca-free) state was predicted to decrease inter-domain separation distance with closure of the SERCA cytoplasmic headpiece. To test this model, we fused Cerulean to the A-domain and YFP to the N or P or TM-domain of SERCA2a. These “2-color” SERCA constructs were expressed in AAV cells, and SERCA structure transitions were detected by changes in intramolecular fluorescence resonance energy transfer (FRET). FRET decreased with thapsigargin for the two N-domain fusion sites (residues 510, 577), while the P- (residue 610) and TM- domain (C-terminus) fusions showed increased FRET with thapsigargin. Unexpectedly, FRET in permeabilized cells was higher in Ca [10 μM] than in EGTA for all constructs, suggesting an increase in domain separation distance with the E1 to E2 transition. These observations were supported by parallel experiments in fluorescence lifetime distribution (FLD) analysis. FLD resolved two broad distributions of fluorescence lifetimes for 2-color SERCA expressed in live cardiac myocytes, consistent with two major FRET states. The relative populations of these states oscillated with electrical pacing, favoring the high FRET (short distance) state during systole (contraction) and the low FRET (long distance) state during diastole (relaxation). We expect 2-color SERCA constructs to be useful for exploring the magnitude, direction, and kinetics of calcium pump conformational changes. They may also be useful for screening candidate compounds for modulation of SERCA pump structure and function.

Assessing the Metabolic State of Cultured Cells and the Intact Cochlea by Two-Photon Fluorescence Intensity and Lifetime Imaging

The ability to match energy production with demand is a fundamental determinant of the health of cells, tissues, and organs of the body. Metabolism and mitochondrial dysfunction have been broadly implicated in many disease states, including hearing disorders and noise-induced hearing loss. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) and flavoprotein (Fp) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, the fluorescence intensity has commonly been used as an indicator of the reduced NADH and/or oxidized Fp concentration in cells and tissue. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. This presents a new opportunity to track the cellular metabolic state as NADH binding changes with metabolism. To better understand what is revealed by variations in the fluorescence intensity and lifetime distributions, we have used mitochondrial uncouplers and inhibitors in conjunction with manipulation of substrate and oxygen concentrations to systematically adjust the metabolic state. We will compare the similarities and differences of the intensity- and FLIM-based assessment of metabolic rate in model systems of increasing complexity, ranging from single cells to intact tissue, highlighting the advantages and limitations of each technique. Supported by NIH DC 02053, NSF-EPSCoR EPS-0701892 (CFD 47.076), NIH P20 RR16469 from the National Center for Research Resources (NCRR), and NIH R15 GM085776.

Automated Analysis of Fluorescence Lifetime Imaging Microscopy (FLIM) Data Based on the Laguerre Deconvolution Method

In fluorescence lifetime imaging microscopy (FLIM), fluorescence time decay at each pixel of the imaged sample are measured. Every recorded fluorescence decay corresponds to the time convolution of the instrument response with the intrinsic fluorescence impulse response function (IRF), from which the sample fluorescence lifetime is determined. To estimate the IRF, the instrument response thus needs to be deconvolved from the recorded fluorescence decay. We have recently introduced a novel FLIM time-deconvolution method based on the linear expansion of the fluorescence decays on an orthonormal Laguerre basis. Since this method allows simultaneous estimation of the IRFs at all pixels, it performs at least two orders of magnitude faster than standard algorithms. In its original implementation, however, the Laguerre basis, determined by the Laguerre parameter α, is selected using a heuristic approach. Here, we present an automated implementation, whereby the Laguerre parameter α is treated as a free parameter within a nonlinear least squares optimization scheme. The new implementation combines the unmatched inherent computational speed of the Laguerre deconvolution method with a systematic model selection approach. This method will thus facilitate applications of FLIM requiring automatic estimation of the spatial distribution of fluorescence lifetimes, such as in in vivo tissue FLIM imaging.

Time- and Spectral-resolved two-photon imaging of healthy bladder mucosa and carcinoma in situ

Combined non-linear imaging techniques were used to deeply image human ex-vivo fresh biopsies of bladder as well as to discriminate between healthy bladder mucosa and carcinoma in situ. Morphological examination by two-photon excited fluorescence and second-harmonic generation has shown a good agreement with corresponding common routine histology performed on the same samples. Tumor cells appeared slightly different in shape and with a smaller cellular-to-nuclear dimension ratio with respect to corresponding normal cells. Further differences between the two tissue types were found in both spectral emission and fluorescence lifetime distribution by performing temporal- and spectral- resolved analysis of fluorescence. This method may represent a promising tool to be used in a multi-photon endoscope, in a confocal endoscope or in a spectroscopic probe for in-vivo optical diagnosis of bladder cancer.