Continuous photodetection of resonance fluorescence

Abstract

The concept of photodetection as a continuous quantum measurement introduced by Srinivas and Davies is extended to the detection of resonance fluorescence. This is a new approach to an old subject. This is also a new development in continuous photodetection in the sense that a fermion rather than the traditional boson field is the subject of monitoring. The superoperators for the no-count and the one-count processes are postulated, following the examples of Srinivas and Davies. The probability for the m-count process is then derived on the basis of these postulates. The first two factorial moments of the photon-number distribution are calculated, which are then used to evaluate the non-classical effects in resonance fluorescence. A parameter slightly different from Mandel's is found to be more suitable indicator of photon antibunching in the transient state.     

Homogeneous immunoassay based on two-photon excitation fluorescence resonance energy transfer

A two-photon excitable small organic molecule (abbreviated as TP-NH 2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared, which emitted fluorescence in the visible region upon excitation at 800 nm. Using the TP-NH 2 molecule as an energy donor, a two-photon excitation fluorescence resonance energy-transfer (TPE-FRET) based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-Cl, a dark quencher) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.     

Homogeneous assay based on anti-Stokes' shift time-resolved fluorescence resonance energy-transfer measurement

We report here a novel, time-resolved, lanthanide-based energy-transfer assay utilizing nonoverlapping acceptor fluorophores, which have their absorption energetically at a higher level than the emittive transitions of the donor. The technique was studied by comparing a series of nonoverlapping acceptors in a homogeneous DNA model assay utilizing Eu3+ chelate as a donor. The assay provides strong energy-transfer enhanced acceptor emission and enables the anti-Stokes' shift FRET measurement, in which the induced acceptor emission is at shorter wavelength than the donor emission. This results in high sensitivity, and 0.8 pM detection limit was measured for the DNA target. The acceptor signal of the assay is characterized by exceptional lifetime properties and is not strictly following the Forster's theory. The mechanism of nonoverlapping energy transfer is considered, and we propose that when nonoverlapping acceptors are utilized, the energy transfer arises from the upper 5D2 and 5D1 excited states of europium. The assumption was studied using a simplified energy level scheme of the Eu3+ donor and the acceptors, and a correlation between the acceptor emission behavior and the energy level scheme was found.     

The asymptotic behaviour of the spectrum of resonance fluorescence

Abstract

The spectrum of resonance fluorescence from laser-driven multi-level systems is investigated and especially its asymptotic frequency dependence in the wings. Analytical results are presented and the transitions within multilevel systems are classified into three categories according to the asymptotic frequency dependence of their spectra. Under certain conditions an arbitrarily fast asymptotic fall-off behaviour of the spectrum is found which, for intermediately strong driving fields, leads to subnatural linewidths. Numerical examples are presented to illustrate the analytical results.     

Influence of buffer on resonance frequency of thermoacoustic engine

Frequency matching is of great importance to a thermoacoustically driven pulse tube refrigeration system. To compute the resonance frequency of thermoacoustic engines, the fluid impedance method is introduced. The calculations of the thermoacoustic engines with different arrangements of buffer have been carried out. The influence of the buffer arrangements and the volume on the resonance frequency as well as the acoustic power of thermoacoustic engines is also discussed.     

Homogeneous noncompetitive immunoassay for 17beta-estradiol based on fluorescence resonance energy transfer

We previously presented a homogeneous noncompetitive assay principle based on quenching of the fluorescence of europium(III) chelate. This assay principle has now been applied to the measurement of 17beta-estradiol (E2) using europium(III) chelate labeled estradiol specific antibody Fab fragment (Eu(III)-Fab) as a donor and E2 conjugated with nonfluorescent QSY21 dye as an acceptor. Fluorescence could be measured only from those Eu(III)-Fab that were bound to E2 of the sample, while the fluorescence of the Eu(III)-Fab that were not occupied by E2 were quenched by E2-QSY21 conjugates. The performance of the assay was tested both in buffer and in the presence of serum. Because approximately half of the Fabs were incapable of binding to E2, the maximum quenching efficiency was only 55%. The lowest limits of detection were 18 and 64 pM in buffer and serum-based calibrators, respectively. The highest measurable concentrations were 0.4 nM in buffer and 1 nM in serum. The presented noncompetitive assay principle requires only one binder, and it can be applied to other haptens as well, providing that a suitable antibody is available.     

Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.     

Influence of fluorescence reabsorption and trapping on solid-state optical cooling

We analyze the random process of fluorescence reabsorption and trapping in solid-state optical materials in general and its influence on the efficiency of optical cooling of solids by anti-Stokes fluorescence in particular. Using the absorption and fluorescence spectra of Yb3+:ZrF4-BaF2-LaF3-AlF3-NaF (ZBLAN) as input data, we employ a random-walk model to test analytical approximations of the fluorescence escape efficiency and cooling efficiency, including reflections at the boundary.     

Plasmon-enhanced resonance Raman scattering and fluorescence in Langmuir-Blodgett monolayers

Localized surface plasmon resonances in silver and gold nanostructures are engaged to enhance the inelastic Raman scattering and the fluorescence of a phopholipid containing a sulforhodamine 101 acid chloride dye known as Texas Red. The most efficient coupling and enhancement are attained when the excitation laser line is in resonance with both the chromophore and the plasmon absorption of the nanostructure, the case of surface-enhanced resonance Raman scattering, allowing single-molecule detection. The tagged phospholipid was incorporated into a single fatty acid Langmuir monolayer at varying concentrations and transferred onto an evaporated Ag nanoparticle film. Surface-enhanced fluorescence is achieved using shell-isolated silica-coated gold nanoparticles, an emission enhancement named SHINEF.     

Fluorescence coincidence spectroscopy for single-molecule fluorescence resonance energy-transfer measurements

Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis of raw burst traces is not always straightforward. The presence of a "zero peak" and the skewness of peaks at high and low FRET efficiencies in proximity ratio histograms make the accurate evaluation of the histogram a challenging task. This is further compounded by the difficulty associated with siting two fluorophores in optimal range of each other. Here we present an alternative method of analysis, based on handling coincident FRET photon bursts, that addresses these problems. In addition, we demonstrate methods to enhance coincidence levels and thus the accuracy of FRET determination: the use of dual-color excitation, including direct excitation of the acceptor fluorophore; the addition of a remote dye to the biomolecule, not involved in the FRET process; or a combination of the two. We show the advantages of dual excitation by studying several labeled double-stranded DNA samples as FRET models. This method extends the application of single-molecule FRET to more complicated biological systems where only a small fraction of complexes are fully assembled.     

Influence of saline media on the fluorescence emission of Bacillus spores

Single-particle levitation in conjunction with 264.3-nm laser excitation is used to measure the fluorescence emission of individual particles of Bacillus globigii spores. With precise humidity control, the fluorescence emission of wetted and desiccated Bacillus spore particles is measured from 300 to 450 nm. Comparison of spectra for Bacillus spores suspended in a standard buffer aqueous solution and for a desiccated 10-mum-diameter aggregate Bacillus spore particle shows that the spectra is virtually indistinguishable. However, at 85% relative humidity, corresponding to a 4.5M sodium chloride solution, the spore spectra redshifts by approximately 25 nm. It is postulated that the spectra redshifting is a result of specific interactions between the tyrosine fluorophore of the Bacillus spore and the phosphate moieties in the buffer solution.     

An efficient and biocompatible fluorescence resonance energy transfer system based on lanthanide-doped nanoparticles

This work demonstrates an efficient and bio-friendly fluorescence resonance energy transfer (FRET) system based on lanthanide-doped inorganic nanoparticles. A facile aqueous route was used to synthesize the CePO(4):Tb nanorods with homogeneous colloidal dispersion, which emits a bright green light with a high quantum yield (～0.36) and a long fluorescence lifetime (～3.50 ms) upon UV excitation. Upon treatment of CePO(4):Tb with aqueous Rhodamine B (RhB), an efficient FRET occurs from the Tb(3+) to the RhB molecules, giving rise to well resolved and ratiometric emissions of donors and acceptors, respectively, with an energy transfer efficiency of up to 0.85. When incubated with HeLa cells at 37?°C, the CePO(4):Tb treated with RhB shows bright intracellular luminescence, indicating that it can be successfully internalized inside the cells and the FRET remains in the living cells. Moreover, the cytotoxic measurements demonstrate good biocompatibility and low cytotoxicity of our present FRET system. The advantages presented above including high quantum yield of donors, high energy transfer efficiency, ratiometric fluorescent emission and good biocompatibility, indicate the high potential of the CePO(4):Tb/RhB FRET system for monitoring biological events.     

Influence of local-site symmetry on fluorescence lifetime in high-Nd-concentration laser materials

We have correlated the radiative fluorescence lifetime of the ${}^4\text{F}{}_{\mathrm{<mtext>3</mtext><mtext>2</mtext>}}$ level of Nd³⁺ in laser crystals, including the recently developed high-Nd-concentration materials such as NdP₅O₁₄, with the local Nd³⁺ site symmetry. At Nd³⁺ concentrations low enough for non-radiative interactions to be negligible, the fluorescence decay rate is determined by the deviation from local inversion symmetry, which admixes even- and odd-parity electronic wavefunctions, allowing ${}^4\text{F}{}_{\mathrm{<mtext>3</mtext><mtext>2</mtext>}}$ radiative decay by electric-dipole transitions.     

Influence of optical properties on two-photon fluorescence imaging in turbid samples

A numerical model was developed to simulate the effects of tissue optical properties, objective numerical aperture (N.A.), and instrument performance on two-photon-excited fluorescence imaging of turbid samples. Model data are compared with measurements of fluorescent microspheres in a tissuelike scattering phantom. Our results show that the measured two-photon-excited signal decays exponentially with increasing focal depth. The overall decay constant is a function of absorption and scattering parameters at both excitation and emission wavelengths. The generation of two-photon fluorescence is shown to be independent of the scattering anisotropy, g, except for g > 0.95. The N.A. for which the maximum signal is collected varies with depth, although this effect is not seen until the focal plane is greater than two scattering mean free paths into the sample. Overall, measurements and model results indicate that resolution in two-photon microscopy is dependent solely on the ability to deliver sufficient ballistic photon density to the focal volume. As a result we show that lateral resolution in two-photon microscopy is largely unaffected by tissue optical properties in the range typically encountered in soft tissues, although the maximum imaging depth is strongly dependent on absorption and scattering coefficients, scattering anisotropy, and objective N.A..     

Tomographic imaging of ratiometric fluorescence resonance energy transfer in scattering media

A method to visualize and quantify fluorescence resonance energy transfer (FRET) in scattering media is proposed. It combines the ratiometric FRET method with fluorescence molecular tomography (FMT) in continuous wave (CW) mode. To evaluate the performance of the proposed method, experiments on a tissue-mimicking phantom are carried out. The results demonstrate that the proposed approach is capable of visualizing and quantifying the FRET distribution in scattering media, which implies the further application of the ratiometric assay in in vivo studies.     

Dendrimer-based nanoprobe for dual modality magnetic resonance and fluorescence imaging

A novel PAMAM dendrimer-based nanoprobe for dual magnetic resonance and fluorescence imaging modalities was synthesized. Fluorescence studies revealed that Gd(III) complexation to the probe has no effect on the quantum yield; however, increases in the dye content resulted in partial quenching. The potential of the new nanoprobe, G6-(Cy5.5)(1.25)(1B4M-Gd)(145), as a dual modality imaging agent was demonstrated in vivo by the efficient visualization of sentinel lymph nodes in mice by both MRI and fluorescence imaging modalities.     

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.     

Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.     

Quantum dot-fluorescent protein pairs as novel fluorescence resonance energy transfer probes

Fluorescence resonance energy transfer (FRET) characteristics, including the efficiency, donor-acceptor distance, and binding strength of six fluorescent protein (FP)-quantum dot (QD) pairs were quantified, demonstrating that FPs are efficient acceptors for QD donors with up to 90% quenching of QD fluorescence and that polyhistidine coordination to QD core-shell surface is a straightforward and effective means of conjugating proteins to commercially available QDs. This provides a novel approach to developing QD-based FRET probes for biomedical applications.