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.     

Method of error analysis for phase-measuring algorithms applied to photoelasticity

We present a method of error analysis that can be applied for phase-measuring algorithms applied to photoelasticity. We calculate the contributions to the measurement error of the different elements of a circular polariscope as perturbations of the Jones matrices associated with each element. The Jones matrix of the real polariscope can then be calculated as a sum of the nominal matrix and a series of contributions that depend on the errors associated with each element separately. We apply this method to the analysis of phase-measuring algorithms for the determination of isoclinics and isochromatics, including comparisons with real measurements.     

Simultaneous two-photon spectral and lifetime fluorescence microscopy

When a fluorescence photon is emitted from a molecule within a living cell it carries a signature that can potentially identify the molecule and provide information on the microenvironment in which it resides, thereby providing insights into the physiology of the cell. To unambiguously identify fluorescent probes and monitor their physiological environment within living specimens by their fluorescent signatures, one must exploit as much of this information as possible. We describe the development and implementation of a combined two-photon spectral and lifetime microscope. Fluorescence lifetime images from 16 individual wavelength components of the emission spectrum can be acquired with 10-nm resolution on a pixel-by-pixel basis. The instrument provides a unique visualization of cellular structures and processes through spectrally and temporally resolved information and may ultimately find applications in live cell and tissue imaging.     

On-chip molecular communication: analysis and design

We consider a confined space molecular communication system, where molecules or information carrying particles are used to transfer information on a microfluidic chip. Considering that information-carrying particles can follow two main propagation schemes: passive transport, and active transport, it is not clear which achieves a better information transmission rate. Motivated by this problem, we compare and analyze both propagation schemes by deriving a set of analytical and mathematical tools to measure the achievable information rates of the on-chip molecular communication systems employing passive to active transport. We also use this toolbox to optimize design parameters such as the shape of the transmission area, to increase the information rate. Furthermore, the effect of separation distance between the transmitter and the receiver on information rate is examined under both propagation schemes, and a guidepost to design an optimal molecular communication setup and protocol is presented.     

Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging

A novel microfluidic approach for the quantification of reaction kinetics is presented. A three-dimensional finite difference numerical simulation was developed in order to extract quantitative kinetic information from fluorescence lifetime imaging experimental data. This approach was first utilized for the study of a fluorescence quenching reaction within a microchannel; the lifetime of a fluorophore was used to map the diffusion of a quencher across the microchannel. The approach was then applied to a more complex chemical reaction between a fluorescent amine and an acid chloride, via numerical simulation the bimolecular rate constant for this reaction was obtained.     

On-chip electro membrane extraction with online ultraviolet and mass spectrometric detection

Electro membrane extraction was demonstrated in a microfluidic device. The device was composed of a 25 μm thick porous polypropylene membrane bonded between two poly(methyl methacrylate) (PMMA) substrates, each having 50 μm deep channel structures facing the membrane. The supported liquid membrane (SLM) consisted of 2-nitrophenyl octyl ether (NPOE) immobilized in the pores of the membrane. The driving force for the extraction was a 15 V direct current (DC) electrical potential applied across the SLM. Samples containing the basic drugs pethidine, nortriptyline, methadone, haloperidol, loperamide, and amitriptyline were used to characterize the system. Extraction recoveries were typically in the range of 65-86% for the different analytes when the device was operated with a sample flow of 2.0 μL/min and an acceptor flow of 1.0 μL/min. With the sample flow at 9.0 μL/min and the acceptor flow at 0.0 μL/min, enrichment factors exceeding 75 were obtained during 12 min of operation from a total sample volume of only 108 μL. The on-chip electro membrane system was coupled online to electrospray ionization mass spectrometry and used to monitor online and real-time metabolism of amitriptyline by rat liver microsomes.     

New splitting algorithms for geometric transformations of digital images and their error analysis

For digital images and patterns under the nonlinear geometric transformation, T: (ξ, η) → (x, y), this study develops the splitting algorithms (i.e., the pixel‐division algorithms) that divide a 2D pixel into N × N subpixels, where N is a positive integer chosen as N = 2 ^k(k ≥ 0) in practical computations. When the true intensity values of pixels are known, this method makes it easy to compute the true intensity errors. As true intensity values are often unknown, the proposed approaches can compute the sequential intensity errors based on the differences between the two approximate intensity values at N and N/2. This article proposes the new splitting–shooting method, new splitting integrating method, and their combination. These methods approximate results show that the true errors of pixel intensity are O(H), where H is the pixel size. Note that the algorithms in this article do not produce any sequential errors as N ≥ N₀, where N₀ (≥2) is an integer independent of N and H. This is a distinctive feature compared to our previous papers on this subject. The other distinct feature of this article is that the true error bound O(H) is well suited to images with all kinds of discontinuous intensity, including scattered pixels. © 2011 Wiley Periodicals, Inc. Int J Imaging Syst Technol, 21, 323–335, 2011     

Multiplex single strand conformation polymorphism analysis by capillary electrophoresis with on-the-fly fluorescence lifetime detection

This paper describes the use of on-the-fly fluorescence lifetime detection (OFLD) for multiplex single strand conformation polymorphism (SSCP) analysis by capillary electrophoresis (CE). The dye labels studied for multiplex SSCP-OFLD-CE analyses included RG, NBD, and BODIPY-FL. The dyes were first investigated for a model system of "Wild Type" and "Mutant" 43-base fragments designed to vary by a single A/T substitution. Two dye pairs, BODIPY-FL/ RG and BODIPY-FL/NBD, were then used to detect the G20210A mutation in the human prothrombin gene. Mobility correction was required for the BODIPY-FL/RG system. Three "blind" analyses were performed of three mixtures that combined a control fragment (wild type-BODIPY-FL) with two "unknown" fragments selected among four possibilities (wild type or mutant labeled with NBD or RG). In each multiplex analysis, the "origin" of the unknown fragments was correctly identified on the basis of fluorescence lifetime of the dye label and the presence or absence of the mutation was correctly determined on the basis of conformation-induced differences in migration time.     

Critical flocculation concentrations, binding isotherms, and ligand exchange properties of peptide-modified gold nanoparticles studied by UV-visible, fluorescence, and time-correlated single photon counting spectroscopies

Protocols for modifying gold nanoparticles with peptide-bovine serum albumin (BSA) conjugates are described within. The resulting constructs were characterized using a number of techniques including static fluorescence spectroscopy and time-correlated single photon counting spectroscopy (TCSPC) in order to quantify peptide-BSA binding isotherms, exchange rates, critical flocculation concentrations, and the composition of mixed peptide-BSA monolayers on gold nanoparticles. TCSPC has proven to be a powerful technique for observing the microenvironment of protein-gold nanoparticle conjugates because it can distinguish between surface-bound and solution-phase species without the need for separation steps. Full characterization of the composition and stability of peptide-modified metal nanoparticles is an important step in their use as intracellular delivery vectors and imaging agents.     

Studying biological tissue with fluorescence lifetime imaging: microscopy,endoscopy, and complex decay profiles

We have applied fluorescence lifetime imaging (FLIM) to the autofluorescence of different kinds of biological tissue in vitro, including animal tissue sections and knee joints as well as human teeth, obtaining two-dimensional maps with functional contrast. We find that fluorescence decay profiles of biological tissue are well described by the stretched exponential function (StrEF), which can represent the complex nature of tissue. The StrEF yields a continuous distribution of fluorescence lifetimes, which can be extracted with an inverse Laplace transformation, and additional information is provided by the width of the distribution. Our experimental results from FLIM microscopy in combination with the StrEF analysis indicate that this technique is ready for clinical deployment, including portability that is through the use of a compact picosecond diode laser as the excitation source. The results obtained with our FLIM endoscope successfully demonstrated the viability of this modality, though they need further optimization. We expect a custom-designed endoscope with optimized illumination and detection efficiencies to provide significantly improved performance.     

Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy

A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 to 575 nm, the emission from 330 to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for 20 (fluorescence lifetime standard/solvent) combinations are given.     

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.     

On-chip, cell-based microarray immunofluorescence assay for high-throughput analysis of target proteins

We have developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, we examined the accumulation of the alpha subunit of the hypoxia-inducible factor (HIF-1alpha) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. We also tested the effect of the known dysregulator of HIF-1alpha, 2-methoxyestradiol (2ME2), on the levels of HIF-1alpha using a dual microarray stamping technique. This chip-based in situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia mimicking conditions and the reduction of HIF-1alpha levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform.     

Depth resolution and multiexponential lifetime analyses of reflectance-based time-domain fluorescence data

Time-domain fluorescence imaging is a powerful new technique that adds a rich amount of information to conventional fluorescence imaging. Specifically, time-domain fluorescence can be used to remove autofluorescence from signals, resolve multiple fluorophore concentrations, provide information about tissue microenvironments, and, for reflectance-based imaging systems, resolve inclusion depth. The present study provides the theory behind an improved method of analyzing reflectance-based time-domain data that is capable of accurately recovering mixed concentration ratios of multiple fluorescent agents while also recovering the depth of the inclusion. The utility of the approach was demonstrated in a number of simulations and in tissuelike phantom experiments using a short source-detector separation system. The major findings of this study were (1) both depth of an inclusion and accurate ratios of two-fluorophore concentrations can be recovered accurately up to depths of approximately 1 cm with only the optical properties of the medium as prior knowledge, (2) resolving the depth and accounting for the dispersion effects on fluorescent lifetimes is crucial to the accuracy of recovered ratios, and (3) ratios of three-fluorophore concentrations can be resolved at depth but only if the lifetimes of the three fluorophores are used as prior knowledge. By accurately resolving the concentration ratios of two to three fluorophores, it may be possible to remove autofluorescence or carry out quantitative techniques, such as reference tracer kinetic modeling or ratiometric approaches, to determine receptor binding or microenvironment parameters in point-based time-domain fluorescence applications.     

Digital micromirror device as a spatial illuminator for fluorescence lifetime and hyperspectral imaging

Time-domain fluorescence lifetime imaging (FLIM) and hyper-spectral imaging (HSI) are two advanced microscopy techniques widely used in biological studies. Typically both FLIM and HSI are performed with either a whole-field or raster-scanning approach, which often prove to be technically complex and expensive, requiring the user to accept a compromise among precision, speed, and spatial resolution. We propose the use of a digital micromirror device (DMD) as a spatial illuminator for time-domain FLIM and HSI with a laser diode excitation source. The rather unique features of the DMD allow both random and parallel access to regions of interest (ROIs) on the sample, in a very rapid and repeatable fashion. As a consequence both spectral and lifetime images can be acquired with a precision normally associated with single-point systems but with a high degree of flexibility in their spatial construction. In addition, the DMD system offers a very efficient way of implementing a global analysis approach for FLIM, where average fluorescence decay parameters are first acquired for a ROI and then used as initial estimates in determining their spatial distribution within the ROI. Experimental results obtained on phantoms employing fluorescent dyes clearly show how the DMD method supports both spectral and temporal separation for target identification in HSI and FLIM, respectively.     

Precision and accuracy of the analog mean-delay method for high-speed fluorescence lifetime measurement

The analog mean-delay (AMD) method is a new alternative method to measure the lifetime of a fluorescence molecule. Because of its powerful advantages of accurate lifetime determination, good photon economy, and a high photon detection rate, the AMD method is considered to be very suitable for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). For the practical usage of the AMD method in FLIM (AMD-FLIM), detailed study on various experimental conditions and parameters that affect the precision and the accuracy of the AMD method is required. In this paper, we present the relation between the precision and accuracy of the lifetime versus iteration number in the AMD method, the best cutoff frequency of a low-pass filter used in the AMD-FLIM system for a given fluorophore, and the optimum position and width of the integration window by using Monte Carlo simulations and a series of AMD-FLIM experiments.     

Elimination of fluorescence and scattering backgrounds in luminescence lifetime measurements using gated-phase fluorometry

A new gated form of phase fluorometry for measuring lifetimes is presented. The technique uses a square-wave excitation and gates the detector on only during the off period of the excitation. Using a long-lived sample, this eliminates or reduces errors from scattered light and short-lived fluorescences. Using a square-wave modulated excitation source with a 50% duty cycle, traditional data treatment can be used after, at most, a simple pi/2 phase adjustment. A combination of theory and experimental results demonstrates the validity of this new gated method and its utility for eliminating or reducing background. The results are precise, accurate, eliminate scattering errors, and greatly reduce errors due to short-lived fluorescence impurities. Errors from fluorescence bleed-through into the detection period or a slow excitation source turn off can be mitigated by using an offset time prior to gating the detector on.     

Crystal structure and fluorescence lifetime of NdAl3(BO3)4, a promising laser material

The structure of NdAl₃(BO₃)₄, determined by single-crystal x-ray analysis, is rhombohedral with space group R32 and cell parameters $\text{a}\text{= 9.3416 (6)}\text{A}\text{?}$, $\text{c}\text{= 7.3066 (8)}\text{A}\text{?}$, Z = 3. A full-matrix, least-squares refinement gives R = 0.033 and R_w = 0.037. The Nd atoms, Al atoms and B atoms occupy trigonal prisms, octahedra, and triangles of oxygen, respectively. Edge-shared Al octahedra form helices along the c-axis. These helices are connected by isolated B triangles and isolated Nd trigonal prisms. The fluorescence lifetime of the ${}^4\text{F}{}_{\mathrm{<mtext>3</mtext><mtext>2</mtext>}}\text{→}{}^4\text{I}{}_{\mathrm{<mtext>11</mtext><mtext>2</mtext>}}$ transition of Nd⁺³ is reported for the system Nd_xGd_1?xAl₃(BO₃)₄. The lifetime of NdAl₃(BO₃)₄ is 19us, and concentration quenching is reduced in the series, as happens in NdNa₅(WO₄)₄ (85μs) and NdP₅O₁₄ (115μs). The shorter lifetime is attributed to noncentrosymmetry; the reduced concentration quenching to isolation of Nd polyhedra.     

On-chip transduction of nucleic acid hybridization using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1.