UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.     

On-the-fly fluorescence lifetime detection of humic substances in capillary electrophoresis

On-the-fly fluorescence lifetime detection was investigated as a tool for studying humic substances in capillary zone electrophoresis (CZE). Humic substances are complex, heterogeneous mixtures of natural products that tend to migrate in a single, broad CZE peak. The intrinsic fluorescence lifetime of five humic substances from the International Humic Substances Society (IHSS) was monitored using excitation at 488 or 364 nm to produce intensity-lifetime electropherograms for each of the substances. Each frequency-domain lifetime measurement, collected at subsecond intervals during the CZE run, contains the equivalent of a complete decay profile. Lifetime analysis of each decay profile was used to construct a lifetime-resolved electropherogram for each lifetime component, from which the variation in relative intensity contributions of each lifetime across the broad CZE peak could be determined. Absorption spectra, fluorescence excitation-emission spectra, and lifetime profiles of batch solutions of the samples were determined as well. It was found that, whereas absorption and fluorescence spectral characteristics tended to discriminate between humic acids and fulvic acids, the batch solution lifetime profiles discriminated instead between samples from different sources, regardless of fraction. On-the-fly lifetime detection provided a more detailed view of the fluorescence decay of the samples, including greater resolution of lifetimes for two of the fulvic acids and greater discrimination among samples based on lifetime profiles across the CZE peaks.     

In situ time-resolved fluorescence spectroscopy in the frequency domain in capillary electrochromatography

In situ time-resolved fluorescence spectroscopy for capillary electrochromatography (CEC) is described in the frequency domain. Fluorescence decay of the solute molecules is collected directly in the packed stationary phase of the CEC capillary. The fluorescence lifetime profile of the solute molecules reveals the microenvironments they experience in the C18 chromatographic interface. A quartz flow cell and experimental optimization of the signal-to-noise ratio are described that enable the collection of high-quality decay data and subsequent calculation of fluorescence lifetime profiles of the solute molecules. The distribution of pyrene (PY), 1-pyrenemethanol (PY-MeOH), and 1-pyrenebutanol (PY-BuOH) into the C18 stationary phase and the solute-C18 phase interactions are probed, under separation conditions for CEC. All three molecules display a Gaussian distribution of lifetimes, consistent with an ensemble of heterogeneous microenvironments in the C18 stationary phase. The least polar molecule PY diffuses deeply into and interacts extensively with the C18 phase, experiencing high hydrophobicity and significant heterogeneity of microenvironments. The retention order of PY-MeOH, PY-BuOH, and PY in CEC is determined by their interactions with the stationary phase, revealed by their fluorescence lifetime distributions.     

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.     

Analysis of heterogeneous fluorescence decays. Distribution of pyrene derivatives in an octadecylsilane layer in capillary electrochromatography

The distribution of solute molecules in the stationary phase in capillary electrochromatography (CEC) has been investigated with time-resolved fluorescence in the frequency domain. The analysis of fluorescence decay poses a challenging problem for the complex decay kinetics of heterogeneous systems such as the C18 stationary phase. The nonlinear least-squares (NLLS) method selects the decay model by minimizing the chi2 value. The chi2 criterion, in conjunction with the requirement that the residues should be randomly distributed around zero, frequently leads to a feasible set of multiple decay models that can all fit the data satisfactorily. The maximum entropy method (MEM) further chooses a unique model from the group of feasible ones by maximizing the Shannon-Jaynes entropy. The unique model, however, is not necessarily the most probable one. In this paper, the best model for the fluorescence decays of solute molecules is selected with NLLS using the chi2 statistics, the stability of the fit, and the consistency within replicate experiments. In addition, the recovered lifetime parameters of the true model should display the same trend as the fluorescence decay profiles when an experimental condition is varied. Using these criteria, a Gaussian distribution of fluorescence lifetimes satisfactorily fits the data under all experimental conditions. An additional minor component with a discrete lifetime is attributed to the systematic errors in the measurements. The distribution is a manifestation of an ensemble of heterogeneous microenvironments in the stationary phase of CEC. MEM is not suitable for the modeling of CEC data because of its inaccuracy in recovering broad fluorescence lifetime distributions and its lack of consistency in the replicate measurements in the studies of high-voltage effects.     

Native fluorescence changes induced by bactericidal agents

Steady-state and time-resolved fluorescence spectroscopy were measured for five species of bacteria (Bacillus subtilis, Staphylococcusaureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa) subjected to three bactericidal agents, formaldehyde, sodium hypochlorite (bleach), and hydrogen peroxide. For all species, the fluorescence was dominated by tryptophan emission with a fluorescence lifetime that can be described by a bi-exponential decay profile. Application of bleach resulted in an almost total loss of scattering and fluorescence, which indicated that total destruction of proteins and amino acids may have occurred. Hydrogen peroxide decreased the fluorescence intensity and shifted /spl lambda//sub max/ to shorter wavelengths, except in S. aureus, which is resistant to oxidizing agents. The formaldehyde shifted /spl lambda//sub max/ to shorter wavelengths for B. subtilis, S. aureus, E. faecalis, and E. coli. The formaldehyde shortened the lifetime of the slow component and increased the amplitude of the fast component relative to the slow component. This study demonstrates that fluorescence spectroscopy offers a method to evaluate the potential for killing bacteria and decontaminating areas by disinfecting agents.     

Nanoparticle-induced fluorescence lifetime modification as nanoscopic ruler: demonstration at the single molecule level

We combine interferometric detection of single gold nanoparticles, single molecule microscopy, and fluorescence lifetime measurement to study the modification of the fluorescence decay rate of an emitter close to a nanoparticle. In our experiment, gold particles with a diameter of 15 nm were attached to single dye molecules via double-stranded DNA of different lengths. Nanoparticle-induced lifetime modification (NPILM) has promise in serving as a nanoscopic ruler for the distance range well beyond 10 nm, which is the upper limit of fluorescence resonant energy transfer (FRET). Furthermore, the simultaneous detection of single nanoparticles and fluorescent molecules presented in this work provides new opportunities for single molecule biophysical studies.     

Photoinduced electron transfer and hole migration along copolymer of vinylbenzenepyrrolidinofullerene[60] and N-vinylcarbazole

Photoinduced electron-transfer and hole-migration processes for newly prepared copolymers of C60 and N-vinylcarbazole (VCz) with the several compositions have been investigated using time-resolved fluorescence and absorption methods. Shortening of fluorescence lifetime of the C60 moiety in PVCz-C60 was observed, suggesting that the charge-separation takes place via the excited singlet state of C60; the rate constants of the charge-separation were evaluated to be larger than 10(9) S(-1) in polar solvents. In the nanosecond transient absorption spectra in the visible and near-IR regions obtained by excitation of the C60 moiety, the generation of PVCz(*+)-C60(*-) was confirmed. The time profiles of these radical ions showed slow decay continuing more than 800 micros in polar solvents. From the simulations of the decay curves on the basis of the proposed kinetic model, in which the charge-recombination takes place competitively with hole-migration, the rate parameters have been evaluated; i.e., the rate constant of hole migration was evaluated to be 5 x 10(7) s(-1), which was 50 times faster than that of the charge-recombination (ca. 10(6) s(-1)) in polar solvent. The charge-recombination takes place after hole migration among 200-300 Cz units along the PVCz chain in PVCz-C60.     

Multispectral fluorescence lifetime imaging of feces-contaminated apples by time-resolved laser-induced fluorescence imaging system with tunable excitation wavelengths

We recently developed a time-resolved multispectral laser-induced fluorescence (LIF) imaging system capable of tunable wavelengths in the visible region for sample excitation and nanosecond-scale characterizations of fluorescence responses (lifetime imaging). Time-dependent fluorescence decay characteristics and fluorescence lifetime imaging of apples artificially contaminated with a range of diluted cow feces were investigated at 670 and 685 nm emission bands obtained by 418, 530, and 630 nm excitations. The results demonstrated that a 670 nm emission with a 418 nm excitation provided the greatest difference in time-dependent fluorescence responses between the apples and feces-treated spots. The versatilities of the time-resolved LIF imaging system, including fluorescence lifetime imaging of a relatively large biological object in a multispectral excitation-emission wavelength domain, were demonstrated.     

An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules

Two procedures based on the weighted least-squares (LS) and the maximum likelihood estimation (MLE) method to confidently analyze single-molecule (SM) fluorescence decays with a total number (N) of 2,500-60,000 counts have been elucidated and experimentally compared by analyzing measured bulk and SM decays. The key observation of this comparison is that the LS systematically underestimates the fluorescence lifetimes by approximately 5%, for the range of 1,000-20,000 events, whereas the MLE method gives stable results over the whole intensity range, even at counts N less than 1,000, where the LS analysis delivers unreasonable values. This difference can be attributed to the different statistics approaches and results from improper weighting of the LS method. As expected from theory, the results of both methods become equivalent above a certain threshold of N detected photons per decay, which is here experimentally determined to be approximately 20,000. In contrast to the bulk lifetime distributions, the SM fluorescence lifetime distributions exhibit standard deviations that are sizably larger than the statistically expected values. This comparison proves the strong influence of the inhomogenuous microenvironment on the photophysical behavior of single molecules embedded in a 10-30-nm thin polymer layer.     

Cross-reactive metal ion sensor array in a micro titer plate format

A cross-reactive array in a micro titer plate (MTP) format is described that is based on a versatile and highly flexible scheme. It makes use of rather unspecific metal ions probes having almost identical fluorescence spectra, thus enabling (a) interrogation at identical analytical wavelengths, and (b) imaging of the probes contained in the wells of the MTP using a CCD camera and an array of blue-light-emitting diodes as a light source. The unselective response of the indicators in the presence of mixtures of five divalent cations generates a characteristic pattern that was analyzed by chemometric tools. The fluorescence intensity of the indicators was transferred into a time-dependent parameter applying a scheme called dual lifetime referencing. In this method, the fluorescence decay profile of the indicator is referenced against the phosphorescence of an inert reference dye added to the system. The intrinsically referenced measurements also were performed using blue LEDs as light sources and a CCD camera without intensifiers as the detector. The best performance was observed if each well was excited by a single LED. The assembly allows the detection of dye concentrations in the nanomoles-per-liter range without amplification and the acquisition of 96 wells simultaneously. The pictures obtained form the basis for evaluation by pattern recognition algorithms. Support vector machines are capable of predicting the presence of significant concentrations of metal ions with high accuracy.     

Picosecond planar laser-induced fluorescence measurements of OH A 2 + ( ' = 2) lifetime and energy transfer in atmospheric pressure flames

A picosecond, excimer-Raman laser (268 nm, 400 ps FWHM) was used for laser sheet excitation of OH in the (2, 0) band. The fluorescence was detected with a fast-gated, intensified camera (400-ps gate width). The effective collisional lifetime of the spectrally integrated fluorescence was measured in two dimensions by shifting the intensifier gate across the decay curve. The average lifetime is ~2.0 ns for a stoichiometric methane -air flame with spatial variations of +/-10 %. Shorter collisional lifetimes were measured for rich flame conditions that are due to a higher number density of the quenchers. Vibrational energy transfer (VET) was observed in premixed methane -air and methane -oxygen flames by putting the fast-gated camera behind a spectrometer. The spectrum of the methane -air flame shows strong VET in contrast with the methane -oxygen flame. This is because N2 is a weak electronic quencher but a strong VET agent. By fitting the measured time dependence of the different vibrational populations ( ' = 2, 1, 0) to a four-level model, rate constants for quenching and VET were determined. For the lower states ( ' = 0, 1) our results are in good agreement with literature values. For a prediction of a spectrally integrated, collisional lifetime in a known collisional environment it is important to consider not only the quenching but also the amount of energy transfer in the excited state as well as the spectral detection sensitivity.     

Time-resolved photoluminescence in Cu(In,Ga)Se2 thin films and solar cells

Room temperature time-resolved photoluminescence (TR-PL) measurements have been performed on Cu(In,Ga)Se₂ (CIGS) thin films and solar cells to clarify the recombination process of the photo-generated minority carrier. Both films and solar cells exhibited PL decay curves composed of the dominant fast (0.7-2 ns) and weak slow (3-10 ns) exponential decay curves. PL lifetime of the cell is longer than that of the thin films, indicating the longer minority carrier lifetime for the hetero-structures than in thin films. The increase of PL lifetime is consistent with the enhancement of the PL intensity and the elimination of defect-related PL as a result of the solar cell formation. These results are discussed in terms of the recombination process of carriers in films and hetero-structures. The relationship between the PL lifetime of the CIGS solar cells and the cell conversion efficiency is described.     

Time-resolved fluorescence spectroscopic study of crude petroleum oils: influence of chemical composition

The fluorescence of crude petroleum oils is sensitive to changes in chemical composition and many different fluorescence methods have been used to characterize crude oils. The use of fluorescence lifetimes to quantitatively characterize oil composition has practical advantages over steady-state measurements, but there have been comparatively few studies in which the lifetime behavior is correlated with gross chemical compositional data. In this study, the fluorescence lifetimes for a series of 23 crude petroleum oils with American Petroleum Institute (API) gravities of between 10 and 50 were measured at several emission wavelengths (450-785 nm) using a 380 nm light emitting diode (LED) excitation source. It was found that the intensity average fluorescence lifetime (tau) at any emission wave-length does not correlate well with either API gravity or aromatic concentration. However, it was found that tau is strongly negatively correlated with both the polar and sulfur concentrations and positively correlated with the corrected alkane concentration. This indicates that the fluorescence behavior of crude petroleum oils is governed primarily by the concentration of quenching species. All the strong lifetime-concentration correlations are nonlinear and show a high degree of scatter, especially for medium to light oils with API gravities of between 25 and 40. The degree of scatter is greatest for oils where the concentrations (wt %) of the polar fraction is approximately 10 +/- 4%, the asphaltene component is approximately 1 +/- 0.5%, and sulfur is 0.5 +/- 0.4%. This large degree of scatter precludes the use of average fluorescence lifetime data obtained with 380 nm excitation for the accurate prediction of the common chemical compositional parameters of crude petroleum oils.     

Conversion of absorbed thermal radiation into visible using europium thenoyltrifluoroacetonate

We evaluate the sensitivity and thermal resolution of the fluorescence of europium (III) thenoyltrifluoroacetonate (EuTTA) in order to convert absorbed thermal radiation into visible. We analyze the variation of the fluorescence properties of EuTTA (specifically, amplitude power and lifetime) after absorbed thermal radiation has caused a change in the local temperature of the material. We propose to analyze the thermal dependence of the fluorescence decay with an integral functional. Such operation correlates the variations of lifetime and amplitude in a single value. With this method of analysis, we study one parameter with increased thermal resolution and linear temperature dependence. The thermal resolution achieved with amplitude power is 0.11 K and with lifetime is 0.83 K at 305 K. The sensitivity of the integral functional is 25 nJ/K, yielding an increased thermal resolution of 0.07 K.     

On the behavior of indole-containing species sequestered within reverse micelles at sub-zero temperatures

We report on the effects of temperature (+30 to -100 degrees C) on the fluorescence from N-acetyl tryptophanamide (NATA) and human serum albumin (HSA) sequestered within Aerosol-OT (AOT) reversed micelles. NATA reports simultaneously from the polar and non-polar side of the reverse micelle interface. As the sample temperature decreases, the relative fraction of NATA molecules associated with the polar side increases. This redistribution process is characterized by DeltaH = -14.8 +/- 0.6 kJ/mol and DeltaS = -54 +/- 2 J/(K mol). The activation energy for thermal quenching (E(a,TQ)) associated with the polar side NATA molecules is 6.7 kJ/mol before the micelles have shed water and 1.0 kJ/mol after water shedding (below approximately -20 degrees C). The time-resolved fluorescence intensity decay for tryptophan-214 in HSA is triple exponential. We suggest that these lifetimes arise from three indole residue conformations in equilibrium. Cooling the sample causes a freezing-in of the least quenched conformer; the other conformers are frozen out. The E(a,TQ) value for the shortest lifetime component is 6 kJ/mol. The E(a,TQ) for the long and intermediate lifetime components are equivalent (approximately 1.5 kJ/mol).     

Fast-gated intensified charge-coupled device camera to record time-resolved fluorescence spectra of tryptophan

The possibilities of a 200 ps gated intensified charge-coupled device (CCD) camera to record time-resolved fluorescence were explored using the fluorescing amino acid tryptophan and its derivative Nacetyl-tryptophan amide (NATA) as model compounds. The results were compared to complementary data from time-correlated single-photon counting (TCSPC) experiments. If a spectral resolution of 1-2 nm is desired, the fast-gated intensified CCD (ICCD) camera is the method of choice. For a 10(-5) M tryptophan solution, time-resolved emission spectra and intensity decays (measured over 12 ns at 25 ps resolution) could be obtained in typically 10 minutes, giving the well-known lifetimes of 0.5 and 3 ns. In addition, a longer lifetime of 7 ns was found at the red edge of the spectrum. The very short gate time of the ICCD camera allowed us to observe a shift in the emission maximum of tryptophan even within the first nanosecond of decay of the fluorescence emission. As expected from the tryptophan rotamer model, such a shift is not observed in NATA. Using amplitudes obtained by global analysis, decay-associated spectra of these lifetimes were constructed.     

Theoretical investigation of the signal-to-noise ratio in fluorescence lifetime imaging

We deduce the signal-to-noise ratio for fluorescence lifetime imaging when using frequency-domain methods. We assume mono-exponential decay and quantum-noise-limited performance. The results are compared with Monte Carlo simulations with good agreement. We also compare our results with previous investigations of time-domain methods for fluorescence lifetime imaging. For a given number of detected photons, we find that frequency-domain and time-domain methods are equally good. The correct choice of detection technique and its parameters is important for obtaining good results.     

Enhanced fluorescence and time resolved fluorescence properties of p-terphenyl crystal grown by selective self seeded vertical Bridgman technique

The fluorescence and fluorescence decay time of a modified Bridgman grown p-terphenyl single crystals have been studied. The fluorescence spectra of selective self seeded vertical Bridgman technique (SSVBT) grown p-terphenyl single crystals exhibit intense peak at 372 nm and a hump at 388 nm. Fluorescence lifetime measured by Time Correlated Single Photon Counting (TCSPC) method for p-terphenyl crystal showed a very short fluorescence decay time (τ) of 3.3 ns.     

Fluorescence decay characteristic of Tm-doped YAG crystal fiber for sensor applications, investigated from room temperature to 1400/spl deg/C

An yttrium aluminum garnet (YAG) crystal fiber with a thulium-doped end tip was specially grown by means of the laser heated pedestal growth approach and designed to be incorporated in a fiber-optic temperature probe. The fluorescence decay characteristics of the crystal fiber, including the temperature dependence of both the fluorescence lifetime and intensity, were comprehensively investigated. Experimental results indicated that the crystal fiber showed a monotonic relationship between the fluorescence lifetime and temperature with an average lifetime sensitivity of 3 /spl mu/s /spl deg/C over a wide temperature range, taking measurement from room temperature to 1200/spl deg/C. Good stability (up to 1400/spl deg/C) was observed with high repeatability of the fluorescence lifetime during the annealing process carried out on the fiber over this temperature range. The fiber was found to be an excellent candidate material to be used as a fluorescence decay-based fiber thermometer probe and the results are presented on its performance.