Significance: Recent evidence suggests that hydroxyapatite (HAP) in sub-retinal pigment epithelial (sub-RPE) deposits in aged human eyes may act to nucleate and contribute to their growth to clinically detectable size. Sub-RPE deposits such as drusen are clinical hallmarks of age-related macular degeneration (AMD), therefore enhanced and earlier detection is a clinical need. We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, can also label the HAP in sub-RPE deposits. However, HAP-bound tetracycline fluorescence excitation and emission spectra overlap with the well-known autofluorescence of outer retinal tissues, making them difficult to resolve.
Aim: In this initial study, we sought to determine if the HAP-bound tetracyclines also exhibit enhanced fluorescence lifetimes, providing a useful difference in lifetime compared with the short lifetimes observed in vivo in the human retina by the pioneering work of Schweitzer, Zinkernagel, Hammer, and their colleagues, and thus a large enough effect size to resolve the HAP from background by fluorescence lifetime imaging.
Approach: We stained authentic HAP with tetracyclines and measured the lifetime(s) by phase fluorometry, and stained aged, fixed human cadaver retinas with drusen with selected tetracyclines and imaged them by fluorescence lifetime imaging microscopy (FLIM).
Results: We found that chlortetracycline and doxycycline exhibited substantial increase in fluorescence lifetime compared to the free antibiotics and the retinal background, and the drusen were easily resolvable from the retinal background in these specimens by FLIM.
Conclusions: These findings suggest that FLIM imaging of tetracycline (and potentially other molecules) binding to HAP could become a diagnostic tool for the development and progression of AMD.
Recently, we discovered microscopic spherules of hydroxyapatite (HAP) in aged human sub-retinal pigment epithelial (sub-RPE) deposits in the retinas of aged humans (PMID: 25605911), and developed evidence that the spherules may act to nucleate the growth of sub-RPE deposits such as drusen. Drusen are clinical hallmarks of age-related macular degeneration (AMD). We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, also stained the HAP spherules, but in general the HAP-bound fluorescence excitation and emission spectra overlapped with the well-known autofluorescence of the RPE overlying drusen, making them difficult to resolve. However, we also found that certain tetracyclines exhibited substantial increases in fluorescence lifetime upon binding to HAP, and moreover these lifetimes were substantially greater than those previously observed (Dysli, et al., 2014) for autofluorescence in the human retina in vivo. Thus we were able to image the HAP spherules by fluorescence lifetime imaging microscopy (FLIM) in cadaveric retinas of aged humans. These findings suggest that FLIM imaging of tetracycline binding to HAP could become a diagnostic tool for the development and progression of AMD.
Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model. These approaches were applied to study the interaction between the Toll Interleukin-1 receptor (TIR) domain of Toll-like receptor 4 (TLR4) and the decoy peptide 4BB. TLR4 was fused to Cerulean fluorescent protein (Cer) and 4BB peptide was labeled with Bodipy TMRX (BTX). Phasor displays for multifrequency FLIM data are presented. The analytical procedure for lifetime background correction is described and the effect of correction on FLIM data is demonstrated. The absolute FRET efficiency was determined based on the phasor plot display and multifrequency FLIM data analysis. The binding affinity between TLR4-Cer (donor) and decoy peptide 4BB-BTX (acceptor) was estimated in a heterogeneous HeLa cell population.
Most of the applications of fluorescence require the use of labeled drugs and labeled biomolecules. Due to the
need of labeling biomolecules with extrinsic fluorophores, there is a rapidly growing interest in methods which provide
label-free detection (LFD). Proteins are highly fluorescent, which is due primarily to tryptophan residues. However,
since most proteins contain tryptophan, this emission is not specific for proteins of interest in a biological sample. This is
one of the reasons of not utilizing intrinsic tryptophan emission from proteins to detect specific proteins. Here, we
present the intrinsic fluorescence for several proteins bound to the silver or aluminum metal nanostructured surfaces. We
demonstrate the metal enhanced fluorescence (MEF) of proteins with different numbers of tryptophan residues. Large
increases in fluorescence intensity and decreases in lifetime provide the means of direct detection of bound protein
without separation from the unbound. We present specific detection of individual types of proteins and measure the
binding kinetics of proteins such as IgG and streptavidin. Additionally, specific detection of IgG and streptavidin has
been accomplished in the presence of large concentrations of other proteins in sample solutions. These results will allow
design of surface-based assays with biorecognitive layer that specifically bind the protein of interest and thus enhance its
intrinsic fluorescence. The present study demonstrates the occurrence of MEF in the UV region and thus opens new
possibilities to study tryptophan-containing proteins without labeling with longer wavelength fluorophores and provides
an approach to label-free detection of biomolecules.
Metal-enhanced fluorescence (MEF) is useful in single molecule detection (SMD) by increasing the photostability,
brightness and increase in radiative decay rates of fluorophores. We have investigated MEF from an individual
fluorophore tethered to a single silver nanoparticle and also a single fluorophore between a silver dimer. The
fluorescence lifetime results revealed a near-field interaction mechanism of fluorophore with the metal particle. Finite-difference
time-domain (FDTD) calculations were employed to study the distribution of electric field near the metal
monomer and dimer. The coupling effect of metal particles on the fluorescence enhancement was studied. We have also
investigated the photophysics of FRET near metal nanoparticles and our preliminary results suggest an enhanced FRET
efficiency in the presence of a metal nanoparticle. In total, our results demonstrate improved detectability at the single
molecule level for a variety of fluorophores and quantum dots in proximity to the silver nanoparticles due to the near-field
metal-fluorophore interactions.
A new method for surface-based fluoroimmunoassays that eliminates separation steps while still allowing high
sensitivity detection of biomolecular interactions is presented. The capture antibody is electrostatically immobilized on a
glass slide coated with a high density silver island film. The metal-enhanced fluorescence generated by the presence of
the islands allows the sensitive detection of bound reporter antibodies versus those free in solution. In order to perform
the measurement, phase-modulation fluorometry is employed which allows observation of the distinct fluorescence
signal of the bound antibodies with a shorter lifetime than unbound antibodies. Here, we show the use of metal-enhanced
fluorescence with phase-modulation fluorometry to quantify monoclonal antibody from a cell culture. The
results show the new technique produces very similar data upon analysis as measured with ELISA analysis. With further
optimization of the procedures, it is forecast that real time monitoring during bioprocessing will be feasible with the
described technique.
Fluorescence is widely used in biological research. Future advances in biology and medicine often depend on the advances in the capabilities of fluorescence measurements. In this overview paper we describe how a combination of fluorescence, and plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology. This change will be based on the use of surface plasmons which are collective oscillations of
free electrons in metallic surfaces and particles. Surface plasmon resonance is now used to measure bioaffinity reactions. However, the uses of surface plasmons in biology are not limited to their optical absorption or extinction. We have shown that fluorophores in the excited state can create plasmons which radiate into the far field; additionally fluorophores in the ground state can interact with and be excited by surface plasmons. These interactions suggest that the
novel optical absorption and scattering properties of metallic nanostructures can be used to control the decay rates, location and direction of fluorophore emission. We refer to this technology as plasmon-controlled fluorescence. We predict that plasmon-controlled fluorescence (PCF) will result in a new generation of probes and devices. PCF is likely to allow design of structures which enhance emission at specific wavelengths and the creation of new devices which control and transport the energy from excited fluorophores in the form of plasmons, and then convert the plasmons back to light.
Silver nanoshells, nanospheres and metal coated dielectric nanowires have been directly compared for their surface enhanced Raman (SERS) activity. SERS was measured from trace deposits of Rhodamine 6G (Rh6G). It was found that the SERS enhancement is largest for samples that are 3D in nature compared to 2D arrays. Furthermore Ag coated dielectric nanowires had the strongest SERS. These results demonstrate the importance of electric field hot spots and molecular orientation in the SERS process.
The interest in optical properties of colloidal metals is driven by their applications to chemistry and optics. Metallic nanoparticles have the ability to enhance the local optical fields and change the spectroscopic properties of organic molecules as well as allow for design new optical devices. Metal-enhanced fluorescence (MEF) is yielding enormous opportunities for enhanced fluorescence sensing and imaging in microfluidics, lab-on-a-chip, clinical diagnostics, microarrays, and cellular applications. MEF is a through-space phenomenon relying on interaction of fluorophores with metallic nano-particles in the presence of excitation light. MEF can be utilized to produce new fluorometric devices with dramatically increased sensitivity.
We report on metal-enhanced fluorescence measured on a silicon surface with silver nanoparticles patterned with electron beam lithography. We employ a combinatorial approach, depositing silver particles ranging in size, shape, inter-particle spacing, and nominal thickness. Two nanoparticle shapes were investigated, square and triangular in cross section with side dimensions ranging from 50 nm to 130 nm and spaced at distances ranging from 150 nm to 390 nm center-to-center. The fluorescence enhancement of several fluorophores was measured with excitation sources consisting of an Ar ion laser at 488/514 nm and a HeNe laser at 633 nm. This approach allows an easy and direct comparison of the fluorescence enhancement to the particle size, shape, inter-particle spacing, and excitation wavelength.
It has been recently shown that the favorable effects of enhanced fluorescent intensities, reduced lifetimes (increased probe photostabilities), enhanced and localized rates of multiphoton excitation, and modified rates of energy transfer can occur for fluorophores or biological species of interest, in close proximity to noble metallic nano-structures and surfaces. Subsequently, nano-metal-enhanced fluorescence (NanoMEF) is yielding enormous opportunities for enhanced fluorescence sensing and imaging in microfluidics, lab-on-a-chip, clinical diagnostics, and cellular applications.
NanoMEF is a through-space phenomenon relying on interaction of fluorophores with metallic nanoparticles in the presence of excitation light. MEF can be utilized to produce nanometer-size sensors, which display enhanced spectral properties, whie still potentially maintaining a probes free space-sensing functionalities. In this presentation we report our recent findings on the effects of silver nano-particles on the spectral properties of two representative fluorescent probes for pH and Ca2+ measurements. We demonstrate that quantum efficiencies of probes are greatly enhanced providing more reliable chemical sensing capabilities. Our findings promise a new class of potential sensors, which we believe could constitute a new breed of composite nanosensors based on metal-enhanced fluorescence and their applications in miniaturized systems.
A luminescent rhenium(I) metal-ligand complex, [Re(bcp)(CO)3(4-COOHPy)](ClO4), where bcp is 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been synthesized and characterized. This complex displays high quantum yields and long excited- state lifetimes (0.3 - 10 microseconds) in fluid solution at room temperature. The metal-to-ligand charge transfer (MLCT) emission from this compound exhibits sensitivity to micro- environment. [Re(bcp)(CO)3(4-COOHPy)]+ displays highly polarized emission with a maximum anisotropy near 0.3 in the absence of rotational diffusion. This Re(I) MLCT complex was conjugated to several biomolecules, including the proteins human serum albumin (HSA) and bovine immunoglobulin G(IgG) as well as an amine containing lipid. When bound to a protein or lipid, the decay time is near 3 microseconds and the quantum yield is approximately 0.12 in aqueous air-equilibrated solution at room temperature. The unique spectral properties and reactive carboxylic acid functionality of [Re(bcp)(CO)3(4-COOHPy)]+, allowed utilization of this probe in numerous biophysical and biomedical applications.
Fluorescence lifetime-based sensing is now recognized as a valuable methodology in clinical and analytical chemistry. For clinical chemistry, or for non-invasive sensing through skin, it is often necessary to obtain quantitative information in highly scattering media. Lifetime-based sensing can be used to obtain quantitative measurements in turbid media. We describe frequency-domain lifetime measurements in intralipid suspension, and present a reliable technique which allows phase-modulation lifetime measurements of fluorophores dispersed within or localized within intralipid. Lifetimes can be measured using an intensity decay law which accounts for the time delays and pulse-broadening effects of multiple light scattering events occurring in the intralipid. Alternatively, the phase and modulation measurements can be performed relative to a reference fluorophore of known lifetime. This approach provided reliable lifetime data for a pH-sensitive fluorophore contained within a microcuvette 4 mm under the surface of an intralipid suspension.
We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. We show that the asymmetric complex Ru(bpy)2(dcbpy) (PF6)2 displays a high anisotropy value near 0.26 when excited in the long wavelength absorption band, and an intensity decay time near 400 ns. For covalent linkage to proteins, we synthesized the N- hydroxy succinimide ester. We measured the intensity and anisotropy decays of [Ru(bpy)2(dcbpy)] when covalently linked to proteins and in solutions of increasing viscosity. These data demonstrated that the probes can be used to measure rotational motions on the microsecond(s) timescale, which so far has been inaccessible using luminescence methods. We also used this probe in a fluorescence polarization immunoassay of HSA. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization. The labeled HSA was also used in a competitive format with unlabeled HSA as the antigen. The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA. These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high molecular weight analytes. The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low molecular weight antigens.
The Center for Fluorescence Spectroscopy (CFS) is a multi-user facility providing state of the art time-resolved fluorescence instrumentation and software for scientists, whose research can be enhanced by such experimental data. The CFS is a national center, supported by the National Center for Research Resources Division of the National Institutes of Health, and in part by the National Science Foundation. Both time-domain (TD) and frequency- domain (FD) measurements (10 MHz to 10 Ghz) are available, with a wide range of excitation and emission wavelengths (UV to NIR). The data can be used to recover distances and site-to-site diffusion in protein, interactions between macromolecules, accessibility of fluorophores to quenchers, and the dynamic properties of proteins, membranes and nucleic acids. Current software provides for analysis of multi-exponential intensity and anisotropy decays, lifetime distribution, distance distributions for independent observation of fluorescence donors and acceptors, transient effects in collisional quenching, phase-modulation spectra and time-resolved emission spectra. Most programs provide for global analysis of multiple data sets obtained under similar experimental conditions. Data can be analyzed on-site by connection with the CFS computers through the internet. During six years of operation we have established scientific collaborations with over 30 academic and industrial groups in the United States. These collaborations have resulted in 63 scientific papers.
We synthesized two conjugatable long-wavelength fluorescence probes. They consist of a squaraine moiety, which is a cyanine- type chromophore with a central squarate bridge and a reactive N- hydroxysuccinimide group for coupling with amino functions. One form is water soluble due to the presence of a sulfobutyl group, the other is water insoluble. The water insoluble form was reacted with taurine to achieve water solubility and this squaraine-taurine conjugate displayed a very high binding affinity to BSA. The squaraines exhibit desirable properties of short lifetimes and low quantum yields in water, with a significant increase of lifetime and quantum yields when bound to proteins. Their absorption maxima around 635 nm in water and 640 nm when bound to proteins allow excitation with the newly commercially available diode lasers sources at 635, 645, and 650 nm. The spectral properties and photostabilities of the water soluble squaraine probes are compared with those of the commercially available CY5-NHS-ester.
Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods are resulting in the rapid migration of time-resolved fluorescence to the clinical chemistry lab, to the patient's bedside, to flow cytometers, to the doctor's office, and even to home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy, and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. In this overview paper we attempt to describe some of the opportunities available using chemical sensing based on fluorescence lifetimes, and to predict those applications of lifetime-based sensing which are most likely in the near future.
Measurements of time-dependent photon migration appear to provide more information for biomedical optical image reconstruction than continuous wave measurements. Yet the ultimate success of photon migration imaging (PMI) for biomedical optical tomography depends upon developing a method which can rapidly measure `time-of-flight' information, and, in near-real time, extract important information required for image reconstruction. Image reconstruction requires information to (1) detect, (2) locate the position and volume, and (3) characterize the optical properties of an optical heterogeneity that would otherwise be obscured by tissue-like scattering. In this presentation, we report PMI `images' of an obscured absorber obtained from two-dimensional time-dependent photon migration measurements which arise from single point source illumination of a scattering medium with modulated light. These PMI `images' along with a theoretical basis for PMI, suggest the potential to rapidly detect and locate the three- dimensional position of an absorber from two-dimensional frequency-domain measurements of phase, (Theta) ((rho) ,f), and modulation, M((rho) ,f). Independent single-pixel measurements and Monte Carlo simulations of (Theta) ((rho) ,f) and M((rho) ,f) confirm the PMI `images' and the hypothesis for PMI.
Measurements of fluorescence lifetimes, rather than intensity or intensity ratios, offer many advantages in clinical chemistry and imaging. However, measurements of time-resolved fluorescence are normally associated with complex laser light sources and instrumentation. In this lecture, we show how emerging technology is enabling the design and use of simple instrumentation for time-resolved fluorescence. In particular, it is now possible to imagine lifetime-based measurements of blood gases and blood glucose, and lifetime imaging of calcium and other ions in microscopic samples.
Chemical sensing with fluorescent probes is usually accomplished by measuring the fluorescence intensity or using wavelength-ratiometric probes. Measurement of fluorescence decay times, rather than intensities, is advantageous because the decay times are largely independent of intensity changes due to light losses, photobleaching, or probe washout. Present technology allows measurement of nanosecond decay times by the phase-modulation method with inexpensive and robust instrumentation. This report describes the use of phase- modulation fluorometry for lifetime-based sensing of O2, pH, Ca2+ and K+.
Fluorescence lifetime imaging (FLIM) is a new methodology in which the image contrast is derived from the fluorescence lifetime, not the local concentration and/or intensity of the fluorophore, at each point in a two-dimensional image. In our apparatus, the lifetime images are created from a series of phase-sensitive images obtained with a gain-modulated image intensifier. The phase-sensitive images obtained with various phase shifts of the gain- modulation signal are used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. Pixel-to-pixel scanning is not required to obtain the images. As an example of biochemical imaging we created lifetime images of the calcium concentration based on Ca2+-induced lifetime changes of calcium green (CaG), which is shown to be highly sensitive to [Ca2+]. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra, which allows Ca2+ imaging using Ca2+ probes which do not display spectral shifts. The concept of fluorescence lifetime imaging has numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence, the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.
We report measurements of site-to-site diffusion in proteins, using frequency-domain measurements of time-dependent energy transfer. The possibility of such measurements is shown from simulations which demonstrate that donor-to-acceptor (D-to-A) diffusion alters the donor frequency response, and that this effect is observable in the presence of a distribution of distances. For decay times typical of tryptophan fluorescence, the simulations indicate D-to-A diffusion coefficients can be measured ranging from 10-7 to 10-5 cm2/s. This possibility was verified by studies of a methylene-chain linked D-A pairs in solutions of varying viscosity. D-to-A diffusion was also measured for acceptor-labeled melittin in the random coil and (alpha) -helical states. Unfolding of troponin I results in increased D-A diffusion. Surprisingly, more rapid diffusion was observed for melittin in the (alpha) -helical state, but over a limited range of distances.
The effect of the collisional quenching on the fluorescence intensity decays has been studied by frequency-domain fluorometry. We used an efficient (CBr4) and/or inefficient (CCl4 quencher to quench the fluorescence of 1,2-benzanthracene (1,2-BA). The wide range of diffusion has been obtained by using propylene glycol at different temperatures (-40 degree(s)C to 40 degree(s)C). The measured intensity decays cannot be satisfactorily fitted either to the Smoluchowski or Collins-Kimball (RBC) model, except the case of inefficient quencher in the presence of high diffusion. In particular, we observed quenching in diffusionless conditions (-40 degree(s)C). To describe the collisional quenching of the fluorescence more correctly we propose a new model which includes a distance-dependent quenching rate (DDQ model). The DDQ simulations show that the local concentration of quencher surrounding the excited fluorophore cannot be approximated by using the RBC model, except in the case of high diffusion and low quenching rate. The DDQ model describes well all measured intensity decays of 1,2-benzanthracene in the presence of CBr4 and/or CCl4. Also, the DDQ model more correctly predicts the curvature of Stern-Volmer plots and activation energies obtained from the temperature dependent rate of quenching.
Measurements of time-resolved fluorescence are increasingly used for research in biophysics, biochemistry, cell biology and medicine. Advances in the technology of light sources and detectors are resulting in more reliable and/or advanced instrumentation, which is resulting in the expanding applications of fluorescence spectroscopy. Time-resolved measurements are often performed by direct measurements in the time-domain. In this article the authors describe the alternative method of frequency-domain fluorometry. The frequency-response of the emission to intensity-modulated excitation can be used to recover the time-dependent decay. Commercial instrumentation now allows measurements to an upper light modulation frequency limit of 200 MHz. This laboratory has developed second and third generation instruments which allows measurements to 2 GHz and subsequently to 10 GHz. The frequency-domain data from such instrumentation provides excellent resolution of picosecond decays of intensity and anisotropy. Additionally, the frequency-domain method appears to provide remarkable resolution of complex decays which are often observed for biochemical samples. In this article the authors describe this instrumentation and applications of this method. Examples are shown using probes with ps decay and correlation times, the intrinsic fluorescence of proteins, and the measurement of end-to-end diffusion in proteins and/or flexible molecules.
We examined the time -dependent donor decays of 2 - amino purine (2 -APU) , in the presence of increasing amounts of acceptor 2-aminobenzophenine (2-ABP). As the concentration of 2-ABP increases, the frequency-responses diverge from that predicted by Forster. The data were found to be consistent with modified Forster equations, but at this time we do not state that these modified expressions provide a correct molecular description of this donor-acceptor system. To the best of our knowledge this is the first paper which reports a failure of the Forster theory for randomly distributed donors and acceptors.
We investigated the influence of end-to-end diffusion on intramolecular energy transfer between a naphthalene donor and dansyl acceptor linked by polymethylene chain. A range of viscosities of 0.6 - 200cP were obtained using propylene glycol at different temperatures (0-80°C) and methanol at 20°C. The intensity decays of naphthalene were measured in frequency-domain. Several theoretical models, including distance distributions were used to fit the data. The results indicate that end-to-end diffusion of flexible donor - acceptor pairs can be readily detected and quantified using frequency-domain fluorometry.
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