Frequency-domain Lifetime Measurements Research Articles

Three-dimensional oxygen concentration monitoring in hydrogels using low-cost phosphorescence lifetime imaging for tissue engineering.

Oxygen concentration measurement in 3D hydrogels is vital in 3D cell culture and tissue engineering. However, standard 3D imaging systems capable of measuring oxygen concentration with adequate precision are based on advanced microscopy platforms, which are not accessible in many laboratories due to the system's complexity and the high price. In this work, we present a fast and low-cost phosphorescence lifetime imaging design for measuring the lifetime of oxygen-quenched phosphorescence emission with 0.25 µs temporal precision and sub-millimeter spatial resolution in 3D. By combining light-sheet illumination and the frequency-domain lifetime measurement using a commercial rolling-shutter CMOS camera in the structure of a conventional optical microscope, this design is highly customizable to accommodate application-specific research needs while also being low-cost as compared to advanced instruments. As a demonstration, we made a fluidic device with a gas-permeable film to create an artificial oxygen gradient in the hydrogel sample. Dye-embedded beads were distributed in the hydrogel to conduct continuous emission lifetime monitoring when nitrogen was pumped through the fluidic channel and changed oxygen distribution in the sample. The dynamics of the changes in lifetime co-related with their location in the gel of size 0.5 mm×1.5 mm×700 µm demonstrate the ability of this design to measure the oxygen concentration stably and precisely in 3D samples.

Fourier Multiplexed Fluorescence Lifetime Imaging.

Fluorescence lifetime imaging microscopy (FLIM) is a widely used functional imaging method in bioscience. Fourier multiplexed FLIM (FmFLIM), a frequency-domain lifetime measurement method, explores the principle of Fourier (frequency) multiplexing to achieve parallel lifetime detection on multiple fluorescence labels. Combining FmFLIM with a confocal scanning microscope allows multiplexed 3D lifetime imaging of cells and tissues. FmFLIM can also be integrated with the scanning laser tomography imaging method to perform 3D multiplex lifetime imaging of whole embryos and thick tissues.

Differential carrier lifetime in InGaN-based light-emitting diodes obtained by small-signal frequency-domain measurements

Recently, a novel method for evaluation of recombination coefficients corresponding to Shockley-Read-Hall, radiative, and Auger recombination channels has been proposed, which combines measurements of the light emitting diode (LED) external quantum efficiency under continuous wave operation with the determination of non-equilibrium carrier differential life time (DLT) by small-signal time-resolved photoluminescence [Nippert et al., Jpn. J. Appl. Phys., Part 1 55, 05FJ01 (2016)]. In this work, we suggest an alternative technique, small-signal frequency-domain lifetime measurements, which is implemented more easily and capable of operating in a wider range of LED operating currents. The DLTs measured by both techniques are shown to agree well with each other, but saturate at low currents, contrary to the trend predicted by the well-known ABC-model. We discuss possible reasons for this deviation, as well as advantages and limitations of the measurement techniques.

Photoluminescence Decay Dynamics in Blue and Green InGaN LED Structures Revealed by the Frequency-Domain Technique

An extended study of charge-carrier localization and delocalization in blue and green InGaN light-emitting diode (LED) test structures has been performed. Using the frequency-domain lifetime measurement (FDLM) technique based on direct harmonic modulation of photoluminescence excitation in the frequency range from 1 Hz to 100 MHz, carrier lifetimes were estimated at scales spanning from milliseconds to nanoseconds. The time resolution was determined using fast Fourier transform analysis. A system comprising a radiative and several nonradiative recombination channels was used to describe the complex photoluminescence decay. Due to the broad timescale, even stretched exponential decays from 2 ns to 4 ns up to 1.5 μs (stretching parameter 0.5 to 0.6) were revealed. A higher degree of carrier delocalization was observed for the blue compared with the green light-emitting structure, providing qualitative insight into disorder, which is tentatively assigned to spatial fluctuations of the indium concentration in the quantum wells. A nanosecond nonradiative recombination channel for the green light-emitting structure was found to be unsaturated throughout the entire photoexcitation power density range and was interpreted as being related to the higher defect density and lower internal quantum efficiency of the sample. To expand the study of lifetimes to much higher photoexcitation power density, time-resolved photoluminescence kinetics were measured.

The effect of pH on the glucose response of the glucose⿿galactose binding protein L255C labeled with Acrylodan

The glucose⿿galactose binding protein (GGBP) is used as an optical biosensor in medical and bioprocess applications. This paper investigates the effect of pH on the behavior of GGBP-L255C labeled with Acrylodan for the purpose of finding the optimum conditions for sensing purposes as well as for protein preparation, purification and storage. The Acrylodan⿿GGBP fluorescence response in absence and presence of glucose was measured under varying buffer and pH conditions. Dissociation constants (Kd) and Gibbs free energies (οG) for the protein-glucose binding were calculated. Binding was found to be energetically favored at slightly acidic to neutral conditions, specifically close to the pI of GBP (⿼5.0). Minimal fluorescence response to glucose was exhibited at pH 3.0 accompanied by a blue shift in the steady state fluorescence spectrum. In contrast, an almost 45% response to glucose was shown at pH 4.5⿿9.0 with a 13-nm red shift. Frequency domain lifetime measurements and quenching with KI suggest that at highly acidic conditions both the glucose-free and the glucose-bound protein are in a conformation distinct from those observed at higher pH values.

PNA-Induced Assembly of Fluorescent Proteins Using DNA as a Framework

Controlled alignment of proteins on molecular frameworks requires the development of facile and orthogonal chemical approaches and molecular scaffolds. In this work, protein-PNA conjugates are brought forward as new chemical components allowing efficient assembly and alignment on DNA scaffolds. Site-selective monomeric teal fluorescent protein (mTFP)-peptide nucleic acid (PNA) (mTFP-PNA) conjugation was achieved by covalent linkage of the PNA to the protein through expressed protein ligation (EPL). A DNA beacon, with 6-Fam and Dabcyl at its ends, acts as a framework to create an assembled hetero-FRET system with the mTFP-PNA conjugate. Using fluorescence intensity, frequency domain lifetime measurements, and anisotropy measurements, the system was shown to produce FRET as indicated by decreased donor intensity, decreased donor lifetime, and increased donor anisotropy. Extension of the DNA scaffold allowed for the assembly of multiple mTFP-PNA constructs. Efficient formation of protein dimers and oligomers on the DNA-PNA frameworks could be shown, as visualized via size exclusion chromatography (SEC) and electrophoresis (SDS-PAGE). Assembly of multiple proteins in a row induced homo-FRET for the mTFP-PNA's assembled on the DNA scaffolds. The oligonucleotide framework allows an induced and controllable assembly of proteins by fusing them to PNAs directed to align on DNA scaffolds.

In Vitro Oxygen Sensing Using Intraocular Microrobots

We present a luminescence oxygen sensor integrated with a wireless intraocular microrobot for minimally-invasive diagnosis. This microrobot can be accurately controlled in the intraocular cavity by applying magnetic fields. The microrobot consists of a magnetic body susceptible to magnetic fields and a sensor coating. This coating embodies Pt(II) octaethylporphine (PtOEP) dyes as the luminescence material and polystyrene as a supporting matrix, and it can be wirelessly excited and read out by optical means. The sensor works based on quenching of luminescence in the presence of oxygen. The excitation and emission spectrum, response time, and oxygen sensitivity of the sensor were characterized using a spectrometer. A custom device was designed and built to use this sensor for intraocular measurements with the microrobot. Due to the intrinsic nature of luminescence lifetimes, a frequency-domain lifetime measurement approach was used. An alternative sensor design with increased performance was demonstrated by using poly(styrene-co-maleic anhydride) (PS-MA) and PtOEP nanospheres.

Improved sensitivity for two-photon frequency-domain lifetime measurement

We demonstrate a method to improve the measurement sensitivity of two-photon frequency-domain lifetime measurements in poor signal to background conditions. This technique uses sinusoidal modulation of the two-photon excitation source and detection of the second harmonic of the modulation frequency that appears in the emission. Additionally, we present the mathematical model which describes how the observed phase shift and amplitude demodulation factor of two-photon phosphorescence emission are related to the phosphorescence lifetime and modulation frequency. We demonstrate the validity of the model by showing the existence of new frequency terms in the phosphorescence emission generated from the quadratic nature of two-photon absorption and by showing that the phase shift and demodulation match theory for all frequency components.

A Magnetically Controlled Wireless Optical Oxygen Sensor for Intraocular Measurements

The influence of oxygen on various ophthalmological complications is not completely understood and intraocular oxygen measurements are essential for better diagnosis and treatment. A magnetically controlled wireless sensor device is proposed for minimally invasive intraocular oxygen concentration measurements. This device will make it possible to make measurements at locations that are currently too invasive for human intervention by integrating a luminescence optical sensor and a magnetic steering system. The sensor works based on quenching of luminescence in the presence of oxygen. A novel iridium phosphorescent complex is designed and synthesized for this system. A frequency-domain lifetime measurement approach is employed because of the intrinsic nature of the lifetime of luminescence. Experimental results of the oxygen sensor together with magnetic and hydrodynamic characterization of the sensor platform are presented to demonstrate the concept. In order to use this sensor for in vivo intraocular applications, the size of the sensor must be reduced, which will require an improved signal-to-noise ratio.

Carrier Lifetimes in GaN Revealed by Studying Photoluminescence Decay in Time and Frequency Domains

The carrier dynamics in GaN epilayer, grown using migration enhanced metalorganic chemical vapor deposition (MEMOCVD), is investigated in a wide range of photoexcitation power density of twelve orders of magnitude. Study of photoluminescence decay lifetimes at extremely low excitation power densities of 0.5 mW/cm2 was performed using frequency-domain lifetime measurements with a UV LED (280 nm) as an excitation source. The carrier lifetimes at high excitation power densities was studied using time-resolved photoluminescence (TR PL) spectroscopy and light-induced transient grating (LITG) technique. The measurements were carried out in the temperature range from 10 to 300 K. At high carrier densities, which are typical for device applications, the room- temperature carrier lifetime of 2 ns was obtained, while a variety of decay components was revealed at low excitation intensities. Trapping and detrapping of the carriers/excitons is shown to play an important role in the carrier dynamics at low carrier densities.

Frequency domain lifetime and spectral imaging microscopy

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.

Effects of Ligands on the Mobility of an Active-Site Loop in Tyrosine Hydroxylase as Monitored by Fluorescence Anisotropy

Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop over the active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, and the three native tryptophan residues were mutated to phenylalanine to form an enzyme with a single tryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzyme resulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did not result in a significant change in the anisotropy in the presence or absence of the deazapterin. The K(d) value for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar results were obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease in anisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes was unaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistent with a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could be modeled as a decrease in the cone angle for the indole ring of about 12 degrees . The data are consistent with a model in which binding of a tetrahydropterin results in a change in the conformation of the surface loop required for proper formation of the amino acid binding site.

Time- and frequency-domain analyses of fluorescence lifetime for temperature sensing

Fluorescence lifetime detection is widely used for sensing physical and chemical quantities. The measurement accuracy of fluorescence lifetime-based sensing systems, either in time or frequency domain relies on their capability of detection and analysis of low level signal superimposed to noise. In this work a quantitative assessment of several data processing and analysis methods for the estimation of the fluorescence lifetime was carried out by using an experimental arrangement based on a fiber optic temperature sensor system. A comparison between the various methods was performed using actual signals from an optical sensing medium. The basic principles of time- and frequency-domain lifetime measurements were also reviewed and discussed in order to point out the limit of the cw frequency-domain approach and to suggest a way to overcome it. The investigated lifetime interval was from 200 to about 2200μs, corresponding to a temperature span of the sensor of about 300 °C. The results showed that in time domain (such as with Marquardt, integration, and log-fit algorithms) a good agreement, with relative differences from 0.2% to 0.5%, can be reached. Frequency-domain results based on an N-point fast Fourier transform (FFT) compare favorably with the previous ones in the long lifetime region (resulting in relative differences lower than 0.2%) with larger differences for short lifetimes. For each data processing method, the uncertainty associated with lifetime estimation was evaluated. Sampling and harmonics effects on the estimation accuracy for N-point FFTs were also investigated to trade-off between speed and accuracy of the algorithm in view of its application in real-time detection systems.

AB‐plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers

Most treatments of frequency domain lifetime measurements indicate that a set of measurements must be made at multiple frequencies in order to determine the lifetimes of the components in a mixture. Although this is the case in general, under special conditions, single-frequency data can resolve multiple lifetimes. Here, data are presented showing several approaches to determining fluorescence lifetimes in two-component mixtures using single-frequency data. Common to all of the procedures presented is exploitation of variations in the relative contributions of a particular fluorophore to the total fluorescence from a mixture of fluorophores. This variation can be produced intentionally by observing a number of samples which contain different relative amounts of the fluorophores. It can be produced fortuitously by observing spatial variations in a mixture of fluorophores in a specimen or set of specimens observed with a lifetime imaging system. It can also be produced by examination of the lifetime spectrum obtained from a fluorophore mixture or by varying the concentration of a quencher in a fluorophore mixture, in which the two fluorophores have different rate constants for quenching. In many instances, the set of approaches presented here will be unsuitable for examination of arbitrary samples of unknown composition for which the multifrequency approach should be used. However, measurements produced using single-frequency methods may be applied to good effect for controlled experiments having defined fluorophores or sets of fluorophores, particularly in the case of biological lifetime imaging studies.

Steady-State and Frequency-Domain Lifetime Measurements of an Activated Molecular Imprinted Polymer Imprinted to Dipicolinic Acid

We recently demonstrated the synthesis and fluorescence activity associated with an optical detector incorporating a molecular imprinted polymer (MIP). Steady-state and time-resolved (lifetime) fluorescence measurements were used to characterize the binding activity associated with MIP microparticles imprinted to dipicolinic acid (DPA). DPA is a unique biomarker associated with the sporulation phase of endospore-forming bacteria. Vinylic monomers were polymerized in a dimethylformamide solution containing DPA as a template. The resulting MIP was then pulverized and sorted into small microscale particles. Tests were conducted on replicate samples of biologically active cultures representing both vegetative stationary phase and sporulation phase of Bacillus subtilis in standard media. Samplers were adapted incorporating the MIP particles within a dialyzer cartridge (500 MW). The permeability of the dialyzer membrane permitted diffusion of lighter molecular weight constituents from microbial media effluents to enter the dialyzer chamber and come in contact with the MIP. Results showed dramatic (10-fold over background) steady-state fluorescence changes (as a function of excitation, emission and intensity) for samples associated with high endospore biomass (DPA), and a frequency-domain lifetime of 5.3 ns for the MIP-DPA complex.

Frequency-domain lifetime fluorometry of double-labeled creatine kinase.

Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of muscle energy metabolism, has been selected for studies of conformational changes that underlie the cellular control of enzyme activity. For fluorescence spectroscopy measurements, the CK molecule was double-labeled with IAF (5-iodoacetamidofluorescein) and ErITC (erythrosin 5'-isothiocyanate). Measurement of fluorescence resonance energy transfer (FRET) from fluorescein to erythrosin was used to obtain information about the donor-acceptor pair distance. Frequency-domain lifetime measurements evaluate the donor-acceptor distance in the native CK molecule as 7.8 nm. The Förster radius equals 5.3 nm with the resolution range from 0.2 to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for artificial conformational changes of the CK molecule with high-salt concentration treatment. The transition distance, defined by His-97 and Cys-283 and derived from a 3D model equals 0.766 nm for the open (inactive) form and 0.277 nm for the closed (reactive) form of the CK molecule. In this way, the resolution range of the used spectroscopy method is significant, concerning the difference of 0.489 nm. Nevertheless, the CK enzyme activity, assessed by the hexokinase-coupled assay, was diminished down to 1 % of the activity of the native enzyme. EFL is suitable for description of conformational behavior implied from the regulation of creatine kinase. However, the observed inhibition restricts EFL to studies of conformational changes during natural catalytic activity.

Real-time background suppression during frequency domain lifetime measurements

We describe real time background suppression of autofluorescence from biological samples during frequency domain or phase modulation measurements of intensity decays. For these measurements the samples were excited with a train of light pulses with widths below 1 ps. The detector was gated off for a short time period of 10 to 40 ns during and shortly after the excitation pulse. The reference signal needed for the frequency domain measurement was provided by a long-lifetime reference fluorophore which continues to emit following the off-gating pulse. Both the sample and the reference were measured under identical optical and electronic conditions avoiding the need for correction of the photomultiplier tube signal for the gating sequence. We demonstrate frequency domain background suppression using a mixture of short- and long-lifetime probes and for a long-lifetime probe in human plasma with significant autofluorescence.

Photostability of Cy3 and Cy5-Labeled DNA in the Presence of Metallic Silver Particles.

We examined the photostability of a double-stranded DNA oligomer, covalently labeled with Cy3 or Cy5 on one strand, in the presence of metallic silver island films. In our experimental configuration a minor fraction of the labeled DNA was close to the silver particles and the remainder was distant from the particles. Proximity of the fluorophores to silver island films resulted in increased intensity. Upon continuous illumination we found a fraction of the emission that was resistant to the photobleaching. The emission spectra of the residual fractions were identical to the initial spectra. The frequency-domain lifetime measurements of this fraction revealed greatly shortened decay times. These results are consistent with the photostable fraction being close to the silver particles. This results suggest that the number of photons detected per fluorophore, prior to photobleaching, can be increased 5-fold or more by proximity to silver particles. Localization at an optimal distance from the silver surface may result in larger enhancements.

Frequency-domain fluorescence microscopy with the LED as a light source.

We describe a frequency-domain lifetime fluorometer based on a microscope and a modulated light-emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz--250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10--15 microm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum-Eu, with a millisecond lifetime, and Ru(bpy)(2)phe-C(12),with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency-domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.

Fluorescence Lifetime Microscopy of the Na+ Indicator Sodium Green in HeLa Cells

This study investigates the usefulness of lifetime measurements of Sodium Green for evaluating intracellular Na+ concentration ([Na+]i) in HeLa cells. Frequency-domain lifetime measurements are performed in HeLa cells and in different buffer solutions (with and without K+ and bovine serum albumin). In all cases, the fluorescence decays of Sodium Green are multiexponential, with decay times independent of [Na+]. Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin results in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponential. The ratio of the fractional intensity of the long decay time (τ2 = 2.4 ± 0.2 ns) to that of the short component (τ1 = 0.4 ± 0.1 ns) increases with [Na+]i. The changes in fluorescence decay with [Na+] are significantly less pronounced in cells as compared with the buffer solutions. Similar values for the resting [Na+]i were estimated from lifetime measurements of Sodium Green and from ratiometric measurements using SBFI. Alternatively, [Na+]i can be monitored by measuring only the phase angle at the modulation frequency of 160 MHz. The usefulness of this latter approach is demonstrated by following the changes in [Na+]i induced by reversible inhibition of the Na+/K+ pump.