Frequency-domain Fluorescence Lifetime Research Articles

ϕ^2FLIM: a technique for alias-free frequency domain fluorescence lifetime imaging

A new approach to alias-free wide-field fluorescence lifetime imaging in the frequency domain is demonstrated using a supercontinuum source for fluorescence excitation and a phase-modulated image intensifier for detection. This technique is referred to as phi-squared fluorescence lifetime imaging (phi(2)FLIM). The phase modulation and square-wave gating of the image intensifier eliminate aliasing by the effective suppression of higher harmonics. The ability to use picosecond excitation pulses without aliasing expands the range of excitation sources available for frequency-domain fluorescence lifetime imaging (fd-FLIM) and improves the modulation depth of conventional homodyne fd-FLIM measurements, which use sinusoidal intensity modulation of the excitation source. The phi(2)FLIM results are analyzed using AB-plots, which facilitate the identification of mono-exponential and multi-exponential fluorescence decays and provide measurements of the fluorophore fractions in two component mixtures. The rapid acquisition speed of the technique enables lifetime measurements in dynamic systems, such as temporally evolving samples and samples that are sensitive to photo-bleaching. Rapid phi(2)FLIM measurements are demonstrated by imaging the dynamic mixing of two different dye solutions at 5.5 Hz. The tunability of supercontinuum radiation enables excitation wavelength resolved FLIM measurements, which facilitates analysis of samples containing multiple fluorophores with different absorption spectra.

Frequency-domain fluorescence lifetime optrode system design and instrumentation without a concurrent reference light-emitting diode

We report the design, development, and implementation of an improved instrumentation approach for frequency-domain fluorescence lifetime (FDFL) optrodic sensing without a concurrent reference LED. FDFL traditionally uses a reference LED, at approximately the same wavelength as the sensor fluorophore emission, to measure phase shifts associated with changes in the fluorescence lifetime of fluorophore. For this work we used an oxygen optrode to design, develop, and test the reference-LED-free FDFL approach. Electronics and optics were optimized, and key system parameters, such as inherent system phase shifts, were determined to insure best performance. In our tests with the oxygen optrode, we observed that several key performance characteristics were improved by the implementation of the reference-LED-free instrumentation platform. This system can potentially be adapted to other analyte-selective fluorophores, which will enable scientists and researchers to expand the application of optrodic sensors as basic research tools in biology, medicine, and agriculture.

Development of Near-Field Fluorescence Lifetime Thermometry

Accurate temperature mapping in nanoscale is essential for the thermal system design of devices. However, conventional optical thermometers cannot achieve nanoscale spatial resolution because of the diffraction limit of light. A novel superresolved optical temperature measurement technique using the near-field fluorescence lifetime is presented. The experimental apparatus based on a frequency domain fluorescence lifetime measurement method has been developed. The fluorophore was molecularly adsorbed onto the sample surface as the nanoscale temperature probe, and the temperature dependence of the fluorescence lifetime was observed. The measurement results of the temperature gradient in a micro wire heater indicate the feasibility of the proposed method.

MhFLIM: Resolution of heterogeneous fluorescence decays in widefield lifetime microscopy

Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring fluorescence lifetimes in widefield microscopy. However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40 MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The system's performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system.

Frequency-Domain Fluorescence Lifetime Measurements via Frequency Segmentation and Recombination as Applied to Pyrene with Dissolved Humic Materials

In this study, the association behavior of pyrene with different dissolved humic materials (DHM) was investigated utilizing the recently developed segmented frequency-domain fluorescence lifetime method. The humic materials involved in this study consisted of three commercially available International Humic Substances Society standards (Suwannee River fulvic acid reference, SRFAR, Leonardite humic acid standard, LHAS, and Florida peat humic acid standard, FPHAS), the peat derived Amherst humic acid (AHA), and a chemically bleached Amherst humic acid (BAHA). It was found that the three commercial humic materials displayed three lifetime components, while both Amherst samples displayed only two lifetime components. In addition, it was found that the chemical bleaching procedure preferentially removed red wavelength emitting fluorophores from AHA. In regards to pyrene association with the DHM, different behavior was found for all commercially available humics, while AHA and BAHA, which displayed strikingly similar behavior in terms of fluorescence lifetimes. It was also found that there was an enhancement of pyrene's measured lifetime (combined with a decrease in pyrene emission) in the presence of FPHAS. The implications of this long lifetime are discussed in terms of (1) quenching mechanism and (2) use of the fluorescence quenching method used to determine the binding of compounds to DHM.

Luminescence Decay Kinetics in GaN Studied by Frequency Domain Measurements

Carrier dynamics in high-quality GaN epilayer was investigated at two extreme excitation levels. Carrier lifetime under high excitation conditions was estimated by using light-induced transient grating technique. Measurements at extremely low excitation power density were performed by using frequency-domain fluorescence lifetime technique. The study was performed in a wide temperature range from 8 to 300 K. The results revealed the influence of donor–acceptor pair recombination and carrier trapping processes.

Frequency Domain Fluorescence Lifetime Study of Crude Petroleum Oils

Frequency domain (FD) fluorescence lifetime data was collected for a series of 20 crude petroleum oils using a 405 nm excitation source and over a spectral range of approximately 426 to approximately 650 nm. Average fluorescence lifetimes were calculated using three different models: discrete multi-exponential, Gaussian distribution, and Lorentzian distribution. Fitting the data to extract accurate average lifetimes using the various models proved easier and less time consuming for the FD data than with Time Correlated Single Photon Counting (TCSPC) methods however the analysis of confidence intervals to the computed average lifetimes proved cumbersome for both methods. The uncertainty in the average lifetime was generally larger for the discrete lifetime multi-exponential model when compared to the distribution-based models. For the lifetime distributions, the data from the light crude oils with long lifetimes generally fit to a single decay term. Heavier oils with shorter lifetimes required multiple decay terms. The actual value for the average lifetime is more dependant on the specific fitting model employed than the data acquisition method used. Correlations between average fluorescence lifetimes and physical and chemical parameters of the crude oils were made with a view to developing a quantitative model for predicting the gross chemical composition of crude oils. It was found that there was no significant benefit gained by using FD over TCSPC other than more rapid data analysis in the FD case. For the FD data the Gaussian distribution model for fluorescence lifetime gave the best correlations with chemical composition allowing a qualitative correlation to some bulk oil parameters.

Fluorescence intensity and lifetime imaging of free and micellar-encapsulated doxorubicin in living cells

Frequency domain fluorescence lifetime imaging microscopy (FLIM) has been used in combination with laser scanning confocal microscopy to study the cellular uptake behavior of the antitumor drug doxorubicin (DOX) and micellar-encapsulated DOX (PLyAd-DOX). The endocytosis uptake process of PLyAd-DOX was monitored over 72 hours using confocal microscopy, with a maximum fluorescence recorded at incubation periods around 24 hours. The micellar structure was not found to release the encapsulated DOX during the time course of imaging. FLIM revealed single lifetime distributions of PLyAd-DOX during accumulation in the cytoplasm. The free DOX in contrast was observed both in the cytoplasm and the nuclear domain of the cell, showing bimodal lifetime distributions. There was a marked dependence of the measured free-DOX lifetime on concentration within the cell, in contrast to reference experiments in aqueous solution, where no such dependence was found. The results suggest the formation of macromolecular structures inside the living cells.

Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy

We investigate the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy, using both theoretical and Monte Carlo methods. Our analysis differs from previous work in that it incorporates the data fitting process used in real experiments, allows for the arbitrary choice of excitation and gain waveforms, and calculates lifetimes as well as associated F-values from higher harmonics in the data. Using our analysis, we found different photon efficiencies to those previously reported and were able to propose optimal excitation and gain waveforms. Additionally, we suggest measurement protocols that lead to further improvement in photon efficiency. We compare our results to other techniques for lifetime imaging and consider the implications of our higher-harmonic analysis for multi-exponential lifetime determination.

Optimizing frequency-domain fluorescence lifetime sensing for high-throughput applications: photon economy and acquisition speed

The signal-to-noise ratio of a measurement is determined by the photon economy of the detection technique and the available photons that are emitted by the sample. We investigate the efficiency of various frequency-domain lifetime detection techniques also in relation to time-domain detection. Nonlinear effects are discussed that are introduced by the use of image intensifiers and by fluorophore saturation. The efficiency of fluorescence lifetime imaging microscopy setups is connected to the speed of acquisition and thus to the imaging throughput. We report on the optimal conditions for balancing signal-to-noise ratio and acquisition speed in fluorescence lifetime sensing.

Segmented Frequency-domain Fluorescence Lifetime Measurements: Minimizing the Effects of Photobleaching Within a Multi-component System

This study introduces a newly developed frequency segmentation and recombination method for frequency-domain fluorescence lifetime measurements to address the effects of changing fractional contributions over time and minimize the effects of photobleaching within multi-component systems. Frequency segmentation and recombination experiments were evaluated using a two component system consisting of fluorescein and rhodamine B. Comparison of experimental data collected in traditional and segmented fashion with simulated data, generated using different changing fractional contributions, demonstrated the validity of the technique. Frequency segmentation and recombination was also applied to a more complex system consisting of pyrene with Suwannee River fulvic acid reference and was shown to improve recovered lifetimes and fractional intensity contributions. It was observed that photobleaching in both systems led to errors in recovered lifetimes which can complicate the interpretation of lifetime results. Results showed clear evidence that the frequency segmentation and recombination method reduced errors resulting from a changing fractional contribution in a multi-component system, and allowed photobleaching issues to be addressed by commercially available instrumentation.

Characterization of biological materials by frequency-domain fluorescence lifetime measurements using ultraviolet light-emitting diodes

Recently developed deep-ultraviolet light-emitting diodes (UV LEDs) emit at wavelengths short enough to excitate fluorescence in most biological autofluorophores. We demonstrate the possibility of harmonical modulation of the output of group-III-nitride based UV LEDs ranging from 255 to 375nm at frequencies up to 200MHz. This enables the application of UV LEDs for frequency-domain fluorescence lifetime sensing with subnanosecond resolution. We report on measurements of fluorescence decay time in common biofluorophores (tyrosine, tryptophan, NADPH, NADH, collagen, DPA, elastin and riboflavin) using commercially available UV LEDs. We demonstrate the capacity of a multichannel LED-based frequency-resolved measurement technique to discriminate between Bacillus globigii and a variety of ambient interferants such as diesel fuel, paper, cotton, dust, etc.

Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo.

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention.

Photoluminescence in sol–gel-derived YAG:Ce phosphors

Cerium-doped yttrium aluminium garnet (Y 3Al 5O 12, YAG) microcrystals were grown using sol–gel technique. The samples were characterized by application of X-ray diffraction (XRD), photoluminescence (PL) spectroscopy and frequency-domain fluorescence lifetime measurements. The PL output, which is important for wavelength conversion in white light emitting diodes (LEDs), was studied as a function of Ce content and annealing temperature. XRD analysis evidenced that sintering of polycrystalline Y 3Al 5O 12:Ce powders at 1000 °C results in the formation of monophasic garnet materials up to 10 mol% of Ce doping; however, concentration quenching is observed in samples with the Ce content exceeding ∼4% probably due to optical losses in the tail states of the imperfect lattice. PL output increased and the concentration quenching disappeared when a higher sintering temperature of 1300 °C was used. However, in high Ce content (>4%) samples, the increase in sintering temperature was found to result in the formation of CeO 2 phase accompanied by an uncontrolled bouncing of PL intensity in powders with similar Ce content.

Combination of a spinning disc confocal unit with frequency‐domain fluorescence lifetime imaging microscopy

Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long acquisition times and more photo bleaching. An alternative is spinning-disc confocal whereby samples are scanned simultaneously by thousands of pinholes, resulting in a virtually instantaneous image with more than tenfold reduced photo bleaching. A spinning disc unit was integrated into an existing FLIM system. Measurements were made of fluorescent beads with a lifetime of 2.2 ns against a 5.3 ns fluorescent background outside the focal plane. In addition, living HeLa cells were imaged with different lifetimes in the cytosol and the plasma membrane. In spinning-disc mode, a lifetime of the beads of 2.8 ns was measured, whereas in wide field a lifetime of 4.1 ns was measured. Lifetime contrast within living HeLa cells could be resolved with the spinning-disc unit, where this was impossible in wide field. Integration of a spinning-disc unit into a frequency-domain FLIM instrument considerably reduces artifacts, while maintaining the advantages of wide field. For FLIM on objects with 3D lifetime structure, spinning-disc is by far preferable over wide-field measurements.

Fluorescence lifetime imaging study of a thin protein layer on solid surfaces

Understanding the fundamental interactions between proteins and solid surfaces is essential in the area of implantable medical devices. Fluorescence methods offer the sensitivity required to study the formation of the initial thin protein layers that mediate biocompatibility of materials. Thin protein layers (bovine serum albumin labelled with 1-anilino-8-naphthalenesulfonate, BSA–ANS) deposited on several surfaces (glass, silicon, stainless steel, polystyrene, and silver island film) were studied using confocal frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) and single-point multifrequency lifetime analysis techniques. FLIM provides spatial information about both fluorophores located on the surface and physicochemical parameters of the surface microenvironment. The average fluorescence lifetimes ( τ av) of the adsorbed BSA–ANS generated by the contact between a protein solution and the material surface were measured by the multifrequency modulation and phase shift. Results indicate that τ av values of the albumin complexes on the surfaces (∼ 12 ns) are, in general, shorter than τ av found in the bulk solution (∼ 14 ns). For some surfaces, like polystyrene and silver island film the differences in τ av of the adsorbed BSA–ANS were found to be much greater. The differences in fluorescence lifetimes may indicate structural changes in the BSA protein induced by contact with the surface.

Calibration of a wide‐field frequency‐domain fluorescence lifetime microscopy system using light emitting diodes as light sources

High brightness light emitting diodes are an inexpensive and versatile light source for wide-field frequency-domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio-frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal-to-noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices ( approximately 40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal-to-noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.

The application of frequency-domain Fluorescence Lifetime Imaging Microscopy as a quantitative analytical tool for microfluidic devices

We describe the application of wide-field frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) to imaging in microfluidic devices. FLIM is performed using low cost, intensity modulated Light Emitting Diodes (LEDs) for illumination. The use of lifetime imaging for quantitative analysis within such devices is demonstrated by mapping the molecular diffusion of iodide ions across a microchannel.

Analysis of heterogeneous fluorescence decays in proteins. Using fluorescence lifetime of 8-anilino-1-naphthalenesulfonate to probe apomyoglobin unfolding at equilibrium

The solvatochromic fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is one of the popular probes of protein folding. Folding kinetics is tracked with ANS fluorescence intensity, usually interpreted as a reflection of protein structure—the hydrophobicity of the binding environments. Such simplistic view overlooks the complicated nature of ANS–protein complexes: the fluorescence characteristics are convoluted results of the ground state populational distribution of the probe–protein complex, the structural changes in the protein and the excited state photophysics of the probe. Understanding of the interplay of these aspects is crucial in accurate interpretation of the protein dynamics. In this work, the fluorescence decay of ANS complexed with apomyoglobin at different conformations denatured by pH is modeled. The fluorescence decay of the ANS–apomyoglobin complex contains information on not only apomyoglobin structure but also molecular populational distributions. The challenge in modeling fluorescence decay profiles originates from the convolution of heterogeneous binding and excited-state relaxation of the fluorescent probe. We analyzed frequency-domain fluorescence lifetime data of ANS-apomyoglobin with both maximum entropy methods (MEM) and nonlinear least squares methods (NLLS). MEM recovers a model of two expanding-and-merging lifetime distributions for ANS–apomyoglobin in the equilibrium transition from the native (N) through an intermediate (I-1) to the acid-unfolded state U A. At pH 6.5 and above, when apomyoglobin is mostly populated at the N-state, ANS–apomyoglobin emits a predominant long-lifetime fluorescence from a relaxed charge transfer state S 1,CT of ANS, and a short-lifetime fluorescence that is mainly from a nascent excited-state S 1,np of ANS stabilized by the strong ANS–apomyoglobin interaction. Lowering the pH diminishes the contribution from the S 1,np state. Meanwhile, more protein molecules become populated at the U A state, which exhibits a short lifetime that is not distinguishable from the S 1,np state. At pH 3.4, when the population of the U A becomes significant, the short-lifetime fluorescence comes predominantly from ANS binding to the U A. Further lowering the pH leads to more exposure of the bound ANS. The long lifetime shifts toward and finally merges with the short lifetime and becomes one broad distribution that stands for ANS binding to the U A below pH 2.4. The above expanding-and-merging model is consistent with F-statistic analysis of NLLS models. The consistency of this model with the knowledge from the literature, as well as the continuity of the decay parameters changing upon experimental conditions are also crucial in drawing the conclusions.