We will overview our initial developments of two, three, and four-photon excitation that started in early nineties with parallel developments in two-color two photon excitation to applications of light quenching (light stimulated emission) as a process to shape the excited state population. We will summarize our basic spectroscopy and time-resolved work in a cuvette system to total internal reflection applications. We will present more recent application of multi-photon processes in the case of metal enhanced fluorescence (MAF) and surface plasmons coupled emission (SPCE). Finally we will present the most recent observation of directly excited phosphorescence (S0→T1 excitation) and discuss potential applications in protein studies and cellular imaging.
Optical microscopes have proven their use as a powerful tool for studying a variety of biological samples. In spite of many successes, there are still numerous obstacles limiting practical applications. Most limiting are the inherent background of physiological samples, photobleaching, and phototoxicity. To allow studies of long lasting processes such as drag delivery, three-dimensional cellular structures, embryogenesis, we have combined a technique called Single Plane Illumination Microscopy (SPIM) with Multi-Pulse Pumping with Time-Gated Detection (MPP-TGD) in order to enhance the signal relative to background. This new method allows for a decrease in light exposure times and improves image quality. This combination allows a new outlook into a variety of important, long-lasting biological processes at a level of detection previously unattainable.
Multi-pulse pumping is a burst of excitation pulses instead of a single pulse which enhances the excited state population of a long-lived label. This label is chosen so that its lifetime is at least 5 times longer than that of typical autofluorescence. The pulse separation within the burst is chosen so that it is at least 5 times shorter than the lifetime of the label. In this case only the population of the fluorescent label is increased and the background remains the same. By subtracting the image acquired with the burst from an image with a single pulse, we were able to increase the signal-to-background ratio of about 100 fold.
Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100- to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.
KEYWORDS: Molecular assembly, Real time imaging, Molecules, Optical microscopy, Image resolution, Ultrafast imaging, Super resolution, In vitro testing, In vivo imaging, Biomedical optics
The long standing unmet need of optical microscopy has been imaging subcellular structures with nanometer precision with speed that will allow following physiological processes in real time. Herein we presenting a new approach (multi-pulse pumping with time-gated detection; MPP-TGD) to increase image resolution and most importantly to significantly improve imaging speed. Alternative change from single pulse to multiple-pulse excitation within continuous excitation trace (in interleave excitation mode) allows for the instantaneous and specific increase (many-folds) in the intensity of subwavelength sized object labeled with long-lived probes. This permits for quick localization of the object. Such intensity change (blinking) on demand can be done with MHz frequency allowing for ultrafast point localization several hundred folds faster than localization based on single molecule blinking. Much higher speed for super-resolution imaging will pave the way for obtaining real time functional information and probing structural rearrangements at the nanometer scale in-vitro and in-vivo. This will have a critical impact on many biomedical applications and enhance our understanding of many cellular functions.
We use the microtubules as a model biological system with our new approach to studying microtubule dynamics in real time. The recent work based on single molecule localization microscopy (SMLM) (Mikhaylova et al., 2015) clearly indicates that microtubules are ~25 nm diameter hollow biopolymers that are organized in a closely spaced (about 20-70 nm apart) microtubule bundles. These structures are organized differently between axons and dendrites and their precise organization in different cell compartments is not completely understood.
In this report, we describe a plasmonic platform with silver fractals for metal enhanced fluorescence (MEF) measurements. When a dye containing surface was brought into contact with silver fractals, a significantly enhanced fluorescence signal from the dye was observed. Fluorescence enhancement was studied with the N-methyl-azadioxatriangulenium chloride salt (Me-ADOTA.Cl) in PVA films made from 0.2 % PVA (w/v) solution spin-coated on a clean glass coverslip. The Plasmonic Platforms (PP) was assembled by pressing together silver fractals on one glass slide and a separate glass coverslip spin-coated with a uniform Me-ADOTA.Cl in PVA film. In addition, we also tested the ADOTA labeled human serum albumin (HSA) deposited on a glass slide for potential PP bioassay applications. Using the new PP, we could achieve more than 20-fold fluorescence enhancement (bright spots) accompanied by decrease in fluorescence lifetime. The experimental results were used to calculate the extinction (excitation) enhancement factor (GA) and fluorescence radiative rate enhancements factor (GF). No change in emission spectrum was observed for a dye with and without contact with fractals. Our studies indicate that this type of PP can be a convenient approach for constructing assays utilizing metal enhanced fluorescence (MEF) without the need for depositing the material directly on metal structures platforms.
Recently, far-field optical imaging with a resolution significantly beyond diffraction limit has attracted tremendous attention allowing for high resolution imaging in living objects. Various methods have been proposed that are divided in to two basic approaches; deterministic super-resolution like STED or RESOLFT and stochastic super-resolution like PALM or STORM. We propose to achieve super-resolution in far-field fluorescence imaging by the use of controllable (on-demand) bursts of pulses that can change the fluorescence signal of long-lived component over one order of magnitude. We demonstrate that two beads, one labeled with a long-lived dye and another with a short-lived dye, separated by a distance lower than 100 nm can be easily resolved in a single experiment. The proposed method can be used to separate two biological structures in a cell by targeting them with two antibodies labeled with long-lived and short-lived fluorophores.
Fluorescent nanodiamonds (NDs) are new and emerging nanomaterials that have potential to be used as fluorescence imaging agents and also as a highly versatile platform for the controlled functionalization and delivery of a wide spectrum of therapeutic agents. We will utilize two experimental methods, TIRF, a relatively simple method based on total internal reflection fluorescence and SPRF, fluorescence enhanced by resonance coupling with surface plasmons. We estimate that the SPRF method will be 100 times sensitive than currently available similar detectors based on detectors. The ultimate goal of this research is to develop microarray platforms that could be used for sensitive, fast and inexpensive gene sequencing and protein detection.
Typically the signal-to-background ratio is the limiting aspect of fluorescence-based detecting and imaging. The background signal can be composed of a variety of sources-excitation scattering, contaminants, and autofluorescence from cellular constituents. Most of these sources have a short-lived lifetime (ps to ns range). In order to increase the signal-to-background ratio, fluorophores with high brightness or in large concentrations are typically used along with time-gated detection. This unfortunately sacrifices the probe’s signal unless it has a very long lifetime. Herein we are presenting a simple method to enhance the detection of widely available and well-known mid-range lifetime (~20 ns) fluorophores’ signal against short-lived backgrounds. This requires a repetition rate of ~300 MHz to pump a 20 ns probe sufficiently. Typical laser sources today are not equipped with repetition rates above 80 MHz. However, this multipulse method allows these rates to be attainable for nearly any pulsed laser source. Multiple pulses of excitation are separated by a variable temporal length, which is short compared to the lifetime of the long-lived fluorophore, to increase the excited state population of a long-lived fluorophore, while the short-lived background decays almost completely between pulses. This is accomplished by simply redirecting the pulsed excitation beam through glass and then a delay length any number of times and lengths as desired to control the number of pulses and separation times.
The over-expression of hyaluronidase has been linked to many types of cancer, and thus we present here a technique for hyaluronidase detection and quantification using Fluorescence Correlation Spectroscopy (FCS). Our probe consists of Hyaluronan macromolecules (HAs) heavily loaded with fluorescein dye to the extent
that the dye experiences self-quenching, and these HAs are detected as very bright, slowly moving particles
by FCS. Hyaluronidase cleaves HAs into HA fragments, increasing the concentration of independent
fluorescent molecules diffusing through the detection volume. The cleavage of HAs releases the self-
quenching so that the intensity of emission is drastically increased. Both the concentration of fluorescent
particles and intensity are measured simultaneously and correlated to the concentration of hyaluronidase. Also, our time correlated system allows us to assess the heterogeneity of the HA solution. Subpopulations of
slowly moving particles with short-lived radiative decay may be separated from fast-moving particles of long-lived radiative decay and studied independently in a technique known as Fluorescence Lifetime Correlation
Spectroscopy (FLCS). Further, we assess the use of the AzaDiOxaTriAngulenium (ADOTA) dye for FCS
experiments. Its lifetime is significantly longer than that of the autofluorescence that plagues fluorescence
experiments involving cells or tissue, and thus the fluorescence decay of the probe can be easily identified and
separated from autofluorescence by FLCS. We demonstrate this by labeling HAs with ADOTA and adding
free Rhodamine 123 to the solution to simulate the autofluorescence. We show that the combination of ADOTA and FLCS allow construction of an FCS-based hyaluronidase assay despite the presence of severe autofluorescence.
Using commercially available organic fluorophores, the current applications of Förster (fluorescence) resonance energy transfer (FRET) are limited to about 80 Å. However, many essential activities in cells are spatially and/or temporally dependent on the assembly/disassembly of transient complexes consisting of large-size macromolecules that are frequently separated by distances greater than 100 Å. Expanding the accessible range for FRET to 150 Å would open up many cellular interactions to fluorescence and fluorescence-lifetime imaging. Here, we demonstrate that the use of multiple randomly distributed acceptors on proteins/antibodies, rather than the use of a single localized acceptor, makes it possible to significantly enhance FRET and detect interactions between the donor fluorophore and the acceptor-labeled protein at distances greater than 100 Å. A simple theoretical model for spherical bodies that have been randomly labeled with acceptors has been developed. To test the theoretical predictions of this system, we carried out FRET studies using a 30-mer oligonucleotide-avidin system that was labeled with the acceptors DyLight649 or Dylight750. The opposite 5′-end of the oligonucleotide was labeled with the Alexa568 donor. We observed significantly enhanced energy transfer due to presence of multiple acceptors on the avidin protein. The results and simulation indicate that use of a nanosized body that has been randomly labeled with multiple acceptors allows FRET measurements to be extended to over 150 Å when using commercially available probes and established protein-labeling protocols.
Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2-3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from localized areas of silver nanostructures.
Scattering from noble metallic nanoparticles with specific structures are strongly depolarized in contrast to
dielectric particles. The effect depends on the shape and symmetry of the nanoparticles and can be explained by
induced plasmonic multi-resonances along different axes of symmetry. In our experiments we found that the
scattering from 'nanorod' structures of silver is more polarized than globular colloidal silver nanostructures. The
depolarized scattering can be tuned to the near-infrared region by using proportionate mixture of the colloids and
nanorods. We demonstrate this effect in solution as well as in polymer films where nanoparticles were
immobilized. This phenomenon of depolarized scattering is promising for designing dye-less sensing devices
useful in diagnostics. We show scattering polarization profile from asymmetric nanostructures changes during
their aggregation. Modulating the rate of aggregation of these nanostructures by 'receptor - ligand'-like interactions
can be successfully utilized for sensitive 'dye-less' diagnostics.
Familial hypertrophic cardiomyopathy (FHC) is a serious heart disease that often leads to a sudden cardiac death of young athletes. It is believed that the alteration of the kinetics of interaction between actin and myosin causes FHC by making the heart to pump blood inefficiently. We set out to check this hypothesis ex vivo. During contraction of heart muscle, a myosin cross-bridge imparts periodic force impulses to actin. The impulses are analyzed by fluorescence correlation spectroscopy (FCS) of fluorescently labeled actin. To minimize observation volume and background fluorescence, we carry out FCS measurements in surface plasmon coupled emission mode in a reverse Kretschmann configuration. Fluorescence is a result of near-field coupling of fluorophores excited in the vicinity of the metal-coated surface of a coverslip with the surface plasmons propagating in the metal. Surface plasmons decouple on opposite sides of the metal film and emit in a directional manner as far-field p-polarized radiation. We show that the rate of changes of orientation is significantly faster in contracting cardiac myofibrils of transgenic mice than wild type. These results are consistent with the fact that mutated heart muscle myosin translates actin faster in in vitro motility assays.
Cerulean and Venus are recently developed fluorescent proteins, often used as a donor-acceptor pair by researchers in Förster resonance energy transfer-based colocalization studies. We characterized the fluorescent properties of these two proteins in a broad spectral range (form ultraviolet to visible region). Excitation spectra, lifetimes, and polarization spectra show significant energy transfer from aromatic amino acids to the fluorescent protein chromophore. High steady-state anisotropy values and the lack of a fast component in anisotropy decays show that the fluorescent protein chromophore is rigidly fixed within the protein structure. Furthermore, we show that the chromophores are not accessible to external quenchers, such as acrylamide or potassium iodide (KI), allowing the removal of "unwanted" background in the environment with external quencher, while leaving the Cerulean/Venus fluorescence unchanged.
Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light.
Metallic particles, silver in particular, can significantly enhance the fluorescence of dye molecules in the immediate
vicinity (5-20 nm) of the particle. This magnifying effect can be theoretically explained/predicted by considering the
change of photonic mode density near the fluorophore due to coupling to the conducting surface. We are using this
method to observe fluorescence from a single ribosomal particle in a project aimed at acquiring sequence information
from the translating ribosome (NIH's $1000 Genome Initiative). Several quartz slides with silver nanostructures were
made using electron beam lithography techniques. The structures were approximately 50 nm high silver tiles measuring
400-700 nm on the side, and were spaced differently over a total area of 1 mm x 1 mm on any given quartz slide. In a
preliminary experiment, we coated this surface with the Alexa 647-labeled antibodies and collected single molecule
images using the MicroTime 200 (PicoQuant) confocal system. We showed that the fluorescence intensity measured
over the silver islands film was more than 100-fold higher than fluorescence from a comparable site on uncoated section
of the quartz slide. No noticeable photobleaching was seen. The fluorescence lifetime was very short, about 200 ps or
less (this is the resolution limit of the system). The method has great promise for investigations of biologically relevant
single molecules.
Recently it has become possible to study single protein molecules in a cell. However, such experiments are plagued by rapid photobleaching. We recently showed that the interaction of fluorophores with localized surface plasmon polaritons (LSPs) induced in the metallic nanoparticles led to a substantial reduction of photobleaching. We now investigate whether the photobleaching could be further reduced when the excited fluorophore interacts with the LSP excited in the metallic nanoparticles resident on mirrored surface. As an example we use myofibrils, subcellular structures within skeletal muscle. We compare nanoparticle-enhanced fluorescence of myofibrils in the presence and in the absence of a mirrored surface. The proximity of the mirrored surface led to enhancement of fluorescence and to a decrease in fluorescent lifetime, much greater than that observed in the presence of nanoparticles alone. We think that the effect is caused by the near-field interactions between fluorophores and LSP, and between fluorophores and propagating surface plasmons (PSPs) produced in the metallic surface by the nanoparticles. Photobleaching is decreased because fluorescence enhancement enables illumination with a weaker laser beam and because the decrease in fluorescence lifetime minimizes the probability of oxygen attack during the time a molecule is in the exited state.
We report new approach to Fluorescence Correlation Spectroscopy (FCS) and Single
Molecule Detection (SMD) based on Surface Plasmon-Coupled Emission (SPCE)
technology. The use of SPCE offers significant reduction of fluorescence volume
(detection volume) reduction decreasing background contribution. Fluorophore
interaction with surface plasmons increases the rate of photon detection and makes
fluorescence very sensitive to change in a position of emitting molecule. The effective
thickness of the fluorescence volume in SPCE experiments depends on two factors: the
depth of evanescent wave excitation and a distance-dependent coupling of excited
fluorophores to the surface plasmons. The excitation with the laser beam at Surface
Plasmon Resonance (SPR) angle (Kretschmann configuration) through the high
numerical aperture objective makes observation volume very shallow below 100 nm. The
layer thickness is further reduced by the metal quenching of excited fluorophores
immediately close to the interface (~10 nm). The fluorescence light is emitted through the
metal film only at the SPCE angle. Any fluorescence occurring at the distances greater
than the coupling distance is effectively reflected (~92%) by the metal film and not
transmitted to the objective. The thickness of the detected volume can be 20-50 nm,
depending on the prism dielectric constants and orientation of the excited dipoles. In
addition the signal is very sensitive to the change in fluorophore position and orientation.
Such strong dependence of the coupling to the surface plasmons on the orientation of
excited dipoles opens new possibilities to study conformational changes of
macromolecules in real time.
Surface plasmon-coupled emission (SPCE) has been used to reduce the detection volume in fluorescence measurements. The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With the excitation through the glass prism at SPR angle (Kretschmann configuration), the detection volume is a composition (product) of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that using Kretchmann configuration in a microscope with high numerical aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL, which is necessary to observe a single cross-bridge in the muscle. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be also used to study the small conformational changes of macromolecules.
We present a novel approach for performing fluorescence immunoassay in serum and whole blood using fluorescently labeled anti-rabbit IgG. This approach, which is based on Surface Plasmon-Coupled Emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the
fluorophores located near a bio-affinity surface. Effective coupling range for SPCE is only couple of hundred nanometers from the metallic surface. Excited fluorophores outside the coupling layer do not contribute to SPCE, and their free-space emission is not transmitted through the opaque metallic film into the glass substrate. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal is discussed. The kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately 2- and 3-fold, respectively (compared to buffer), resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Both glass and plastic slides can be used for SPCE-based assays. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood, without any need for washing steps.
We present a new method for multi-color fluoroimmunoassays based on directional surface plasmon-coupled emission (SPCE). SPCE is coupling of excited fluorophores with a nearby thin metal film (silver) resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagates through the prism depends on emission wavelength and makes possible measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using two antibodies labeled with different fluorophores, binding to an antigen protein immobilized on the silver surface. We observed independent emission at a different angle on the glass prism, resulting of the surface binding of each antibody. This methodology can be readily extended to 3 or more fluorophores. This technology presents opportunity to develop highly sensitive multiplex assay format for biological agents' detection.
We have recently shown that metallic particles or colloids when deposited on the transparent surface can enhance fluorescence properties of nearby fluorophores. We obtained the fluorophore-metal colloid complexes that display significant fluorescence signal enhancement in solution.
Silver nanoparticles (about 20-50 nm size) were synthesized as a stable yellow colloidal solution, and coated with proteins labeled with fluorophores. A several-fold amplification of the fluorescence signal in presence of colloid Ag nanoparticles in solution was observed. Such fluorophore-metal complex presents a unique opportunity for developing of new class of contrast agents for optical imaging and fluorescence based sensing. Solution of silver nanoparticles with enhanced fluorescence can be used in various assays such as DNA hybridization or immunoassays for high sensitivity detection.
We describe the development of a novel generic approach to fluorescence sensing based on metal-enhanced fluorescence (MEF). This work follows our initial reports of radiative decay engineering (RDE), where we experimentally demonstrated dramatic signal enhancements of fluorophores positioned close to surface-bound silver nanostructures. The attractive changes in spectral properties of fluorophores includes increased rates of excitation, increased quantum yields, decreased fluorescence lifetimes with an increased photostability, and drastically increased rates of multi-photon excitation. In this report we present a new class of fluorescent biomarkers which are strongly enhanced by metallic particles. This has afforded the development of a novel generic approach for ultra-sensitive fluorescence assay technology. The assay platform utilizes metal particles deposited on glass/quartz surfaces, covered with sub-nanometer layers of a fluorescent biomarker. As such the fluorescence signal of the composite is strongly enhanced. This readily allows easy, quantitative and inexpensive fluorescence detection of minimal traces of specific antigens. We also explore different sensing geometries, such as using evanescent wave excitation.
We described a new approach to measuring DNA hybridization using surface plasmon-coupled emission (SPCE). This phenomenon occurs for fluorophores within few hundreds of nanometers of a thin metal film on a glass substrate, in our case a 50 nm thick silver film. Excited fluorophores coupled with the surface plasmons in the metal resulting in directional emission through the glass substrate. We studied the emission of Cy3-labeled DNA oligomers bound to complementary unlabeled biotinylated-oligomers, which were bound to the metal surface via a streptavidin-BSA monolayer. Hybridization resulted in directional emission of Cy3-DNA into the prism. Additionally, the use of SPCE resulted in suppression of interfering emission from non-complementary Cy5-DNA oligomers due to weaker coupling of the more distant fluorophores with the surface plasmons. A large fraction of the total potential emission can couple to the surface plasmon resulting in improved sensitivity. We expect SPCE to have numerous applications to nucleic acid analyses.
KEYWORDS: Quantum efficiency, Luminescence, Energy transfer, Energy efficiency, Resolution enhancement technologies, Absorption, Tissues, Fluorescence spectroscopy, Microscopy, Resonance energy transfer
Fluorescence detection is the dominant technology for cellular imaging, clinical diagnostics,
DNA analysis and drug discovery. In fluorescence microscopy, the fluorophores are subject to
photobliching and detectability is limited by cellular autofluorescence. We describe new approach to
develop fluorescent probes that display long emission wavelength, long decay times, and high quantum
yield and high fluorescence brightness. These luminophores are covalently linked pairs of long-lifetime
fluorophores (like metal-ligand complexes) and a short-fluorescence-lifetime and high quantum yield
dyes. Using resonance energy transfer (RET) it is possible of obtaining desirable spectral properties and
long fluorescence lifetime in covalently linked pairs. The long-lifetime donor results in a long-lived
fluorescence component in the acceptor decay. Importantly the emission spectrum of the luminophore
is that of the acceptor and quantum yield of the luminophore approaches that of the higher quantum
yield acceptor. Such luminophores are suitable for fluorescence measurements in biological samples
with the use of real time background suppression to eliminate autofluorescence.
We discuss experimental examples based on long lived metal-ligand-complexes and long
wavelength acceptors like Texas Red. The emission maxima (spectra) and decay time of such RET
tandems can be readily adjusted by selection of the donor, acceptor and distance between them. Such
luminophores with long-wavelength emission and adjustable long lifetime can have numerous
applications in one-photon and multi-photon cellular and tissue imaging with the use of off-gating the
excitation pulse and sample autofluorescence.
In this presentation we describe a novel methodology for ultra-sensitive fluorescence immunoassays based on a new class of fluorescent biomarkers, which are strongly enhanced by nano-size metallic particles. Specifically, we discuss development of the immunoassay on the surfaces coated with metallic particles for high sensitivity detection of cardiac markers. This technology will allow detection of the biomarkers in serum and blood without separation and amplification steps. We present an experimental platform that uses front-face excitation in total internal reflection mode for efficient rejection of background fluorescence.
Directional fluorescence emission of a sulforhodamine 101 in polyvinyl alcohol film has been observed from samples deposited on semi-transparent silver mirror. The fully p-polarized fluorescence emerges through the glass prism in form of hollow cone. The angle of this cone of emission depends on the thickness of the sample, and does not depend on the mode of excitation. The angular dependence of surface plasmon-coupled emission (SPCE) on the sample thickness has been discussed as well as its relevance to the surface plasmon resonance (SPR) analysis.
We report recent achievements in metal-enhanced fluorescence. Several fluorophore systems have been studied on metal particle-coated surface and in colloid suspensions. In particular, we describe a distance dependent enhancement on silver island films (SIFs), release of self-quenching of fluorescence near silver particles, and the applications of fluorescence enhancement near metalized surfaces to bioassays. We discuss a number of methods for various shpae silver particle deposition on surfaces.
We have recently observed both one and multicolor luminescent blinking from a variety of noble-metal
nanostructures upon laser-light illumination. Our results suggest a new class of metallic probes, based on intrinsic metal
luminescence, with several advantages over conventional organic fluorophores, such as enhanced photostability, higher
luminescence intensity, tunable emission wavelengths and the possibilities for a variety of functional surface chemistries.
Our findings for silver, gold and copper nanostructures have revealed some notable and attractive differences in their
individual luminescence properties.
Fluorescence spectroscopy is a widely used research tool in biochemistry and has also become the dominant method enabling the revolution in medical diagnostics, DNA sequencing and genomics. In this forward-looking article we describe a new opportunity in fluorescence, radiative decay engineering (RDE). By RDE we mean modifying the emission of fluorophores or chromophores by a nearby metallic surface, the most important effect being an increase in the radiative decay rate. We describe the usual effects expected form increase in the radiative rates with reference to the biomedical applications of immunoassay and DNA hybridization. We also present experiments which show that metallic particles can increase the quantum yield of low quantum yield fluorophores, increase fluorophore photostability and increase the distance for resonance energy transfer. And finally we show that proximity to silver particles can increase the intensity of the intrinsic fluorescence from DNA.
We describe a new approach to fluorescence sensing based on fluorescence polarization measurements. The sensing device consists of an analyte sensitive fluorescence probe and a reference fluorophore which is not affected by the analyte. Combined emission from probe and reference passes through two adjacent orthogonally oriented polarizers and is viewed with second analyzer polarizer. Changes in the probe intensity result in changes in the polarization of the combined emission. The analyzer polarizer is rotated to yield equal intensity from both sides of two orthogonally oriented polarizers. So constructed sensor earlier was used for manual visual detection of RhB in intralipid and to measure pH using 6-carboxyfluorescein. A sensor equipped with the simple electronic detection system of a dual photocell and a Watson bridge improves the accuracy. We used this device with UV hand lamp, electroluminescent light source or LED to detect pH, oxygen and calcium. This sensing method is generic and can be used with any fluorophore which displays an analyte-dependent change in intensity.
We describe a new method for fluorescence sensing based on measurements of the steady state polarization of an analyte- sensitive fluorophore in the presence of a reference fluorophore with known polarization. The basic concept is that the polarization of a mixture reflects a weighted average of the polarization of the emitting species. By use of reference fluorophores the starting values can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the sensing fluorophore due to the analyte result in changes in the polarization of the combined emission. A wide dynamic range is available because of the freedom to select high or low starting polarization values. Polarization-based sensing was demonstrated for pH using 6- carboxy fluorescein. We also show that polarization sensing can be used for measurements of oxygen and glucose. Polarization sensing can have numerous applications in clinical and analytical chemistry.
We devised an optical assay for glucose based on the genetically-engineered glucose/galactose binding protein (GGBP) from E. coli and phase-modulation fluorometry. A single cysteine mutation was introduced at position 26 of GGBP. When labeled with the sulfhydryl-reactive probe I-ANS, GGBP showed a more than 50% decrease in florescence intensity with increasing glucose concentration (Kd approximately 1 (mu) M). This is consistent with the glucose-bound structure of GGBP where residue 26 becomes more exposed to the aqueous media. Since minimal lifetime changes were observed with glucose binding, a modulation sensor was devised wherein a long lifetime ruthenium metal-ligand complex (Ru) was painted on the surface of the cuvette containing ANS26-GGBP. Glucose binding resulted in changes in the relative intensities of ANS26-GGBP and Ru which were observed as dramatic changes in the modulation at a low frequency of 2.1 MHz. The modulation measured at 2.1 MHz accurately determines the glucose concentration to plus or minus 0.2 (mu) M.
The increasing availability of fs lasers has resulted in expanded interest in multi-photon excitation of fluorescence. In this overview paper, we describe recent developments in multi-photon induced fluorescence spectroscopy and anisotropy. Experimental results are presented for three-photon excitation of tyrosine and tryptophan, and for the single tryptophan protein, troponin C Mutant F22. Three-photon excitation has been also applied to study DPH-labeled membranes and DNA stained with DAPI. The enhancement in spatial resolution resulting from three-photon excitation in spectroscopic and microscopic conditions is described for BBO scintillator in solution. We also describe two-photon excitation with using two-photon at different wavelengths.
We demonstrated that fluorescence anisotropy can be effectively decreased or increased in the presence of light quenching, depending on relative polarizations of excitation and quenching pulses. For parallel light quenching anisotropy decreases to 0.103 and z-axis symmetry is being preserved. In the presence of perpendicular light quenching, the steady- state anisotropy of pyridine 2 glycerol solution increases from 0.368 for unquenched sample to 0.484, for quenched one. We show that angular distribution of transition moments loses the z-axis symmetry in the presence of perpendicular light quenching. In these cases we used more general definitions of anisotropy. Induced by light quenching anisotropy can be applied in both, steady-state and time-resolved measurements. In particular, the systems with low or none anisotropy can be investigated with proposed technique.
Hycamtin is a camptothecin anticancer analogue containing a dimethylaminomethyl substituent at position 9 and a hydroxy functionality at position 10. Using an excitation wavelength of 800 nm we have compared the two-photon cross sections and excited-state lifetimes from several camptothecins in phosphate buffered saline solution with and without the presence of human serum albumin (HSA). Drug and HSA concentrations of 10 (mu) M and 46 (mu) M were employed in our studies. In phosphate buffered saline solution containing HSA the following excited-state lifetimes (ns) and two- photon cross-sections (10-50 cm4 s/photon), respectively, were determined: hycamtin (4.3 nm, 36); camptothecin (1.3 ns, 1); 7-t-butyldimethylsilyl-10- hydroxycamptothecin (1.7 ns, 3.7); 7-t-butyldimethylsilyl- camptothecin (1.9 ns, 1.9); 7-trimethylsilyl-10- aminocamptothecin (6.3 ns; 35); and 7-trimethylsilyl-10- hydroxycamptothecin (1.8 ns; 2.2). Our results indicate that Hycamtin exhibits a high cross-section relative to the parent camptothecin molecule and represents one of the best camptothecin analogues to detect using two-photon excitation. Hycamtin was detected at concentrations as low as 0.05 (mu) M and 1 (mu) M in plasma and whole blood, respectively. The newly synthesized analogue 7- trimethylsilyl-10-aminocamptothecin was found to display similar lifetime and two-photon cross section values relative to Hycamtin. Thus, fluorescence detection with two- photon excitation may prove to be of advantage in the development of this promising new experimental therapeutic.
Recent FDA-approval of topotecan (9-dimethylaminomethyl-10- hydroxycamptothecin) and camptosar (CPT-11) along with the accelerated clinical development of related camptothecin drugs provides new hope for the successful treatment of human cancer, including neoplasms for which no effective treatments currently exist. Current clinical efforts worldwide are aimed at optimizing the therapeutic efficacies of the camptothecins, with the major focus on the determination of the most effective dosing schedules. To this end, technological advances which provide a direct and rapid means of measuring plasma drug levels (i.e. such that correlations between plasma drug levels and clinical responses can be sought) would be of great utility. Here we report on the direct fluorescence detection of topotecan and SN-38 in human plasma and topotecan in whole blood at micro molar levels using two-photon excitation at 730 or 820 nm. Topotecan was detected at concentrations as low as 0.05 and 1 (mu) M in plasma and whole blood, respectively. Since skin, blood and other tissues are translucent at long wavelengths, our results suggest the attractive possibility of homogeneous or noninvasive clinical sensing of camptothecins in situ using two-photon excitation.
We observed fluorescence emission from p-terphenyl, 2,5- diphenyl-1,3,4-oxadiazole (PPD) and indole resulting from two-photon excitation with two different wavelengths near 380 and 760 nm. For two-color two-photon (2C2P) excitation the emission spectra and intensity decays were the same as observed with single photon excitation with an equivalent energy near 250 nm. The two-color two-photon induced emission was observed when the samples were illuminated with both wavelengths, but only when the ps laser pulses were spatially and temporally overlapped. The signals were typically 50-fold to 1000-fold less for illumination at 380 or 760 nm alone. When illuminated with both wavelengths, and when both beams were simultaneously attenuated to the same extent, the emission intensity depended quadratically on the total illumination power, indicating two-photon excitation. When the illumination intensity at one wavelength was attenuated, the signal depended linearly on the power at each wavelength, indicating the participation of one-photon at each wavelength to the excitation process. For 2C2P excitation with both beams vertically polarized the time- zero anisotropies were larger than possible for single photon excitation. For PPD and p-terphenyl with intensity depended on the polarization of each beam in a manner consistent with co-linear transitions, but more complex behavior was found for indole. These results demonstrate that two-color two-photon excitation can be readily observed with modern ps laser sources. This phenomenon can have numerous applications in the chemical and biomedical sciences, as a method for spatial localization of the measured volume.
The use of light quenching to selectively eliminate the emission of biochemical fluorophores based on the
emission wavelength is described. To demonstrate the possibility of wavelength-selective light quenching, a
mixture of two fluorophores, 4-(dimethylamino)-4'-cyanostillene (DCS) and Prodan, emitting at different
wavelengths was examined first. The emission spectrum and intensity decay were altered by the 570-nm quenching pulse due to selective quenching of the longer wavelength emission of DCS. Quenching of the
solvent-sensitive fluorophore partially bound to human serum albumin and partially in the aqueous phase
was then examined. Light quenching with a long wavelength (570 nm) time-delayed pulse selectively
quenched the Prodan fluorophore in the aqueous phase while in the presence of the Prodan bound to human
serum albumin, which emitted at shorter wavelengths, was not quenched. Using one-beam short wavelength
excitation and quenching, a selective quenching of the blue-shifted emission of ribonuclease T1 in the presence of the red-shifted emission of the tryptophan residue in adrenocorticotropic hormone was observed. In both systems wavelength-selective light quenching was demonstrated by a shift in the emission spectra, and by changes in the intensity decay consistent with preferential quenching of one species. Light quenching is instantly reversible by blocking or defocusing of the quenching beam. It can occur for inaccessible residues or in viscous solvents, and thus can be of wide applicability for resolving the complex emission of biological macromolecules.
Three-photon excitation of 2,5-bis(4-biphenyl) oxazole (BBO) was observed when it was excited with the fundamental output of a femtosecond Ti:sapphire laser above 820 nm. The emission spectrum of BBO was
identical for one-, two-, and three-photon excitation at 320, 640, and 960 nm, respectively. In toluene and triacetin, the emission intensity of BBO depended on the square of the laser power for wavelengths below 820 nm and displayed a sharp transition to a cubic dependence at longer wavelengths. The spatial distribution of the emission of BBO with three-photon excitation was more strongly localized than for two-photon excitation of a coumarin fluorophore at the same wavelength. The same single exponential intensity decay was observed for one-, two-, and three-photon excitation. However, the frequency domain anisotropy decay with threephoton excitation at 960 nm revealed a larger time-zero anisotropy, larger differential polarized phase angle, and larger modulated anisotropy than is possible for two-photon excitation with colinear oscillators. In triacetin, the anisotropy is not constant for three-photon excitation at different wavelengths. Surprisingly, the
fluorescence intensities for three-photon excitation were only about 100-fold less than for two-photon excitation. The increasing availability of Ti:sapphire lasers suggests that multiphoton excitation can become a common tool in fluorescence spectroscopy.
We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.
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 studied the steady state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross-section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 nm and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for one- and two-photon excitation. The anisotropy decay measurements of bis-benzimide, 2,5'-bi-1H-benzimidazole, 2'-(4- ethoxphenyl)-5-(4-methyl-1-piperazinyl) (HOECHST 33342) in ethanol revealed the same correlation times for two-photon excitation as observed for one-photon excitation. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for two-photon excitation than for one-photon excitation. The anisotropy spectra of Hoechst 33342 was examined in glycerol at -20 degree(s)C, revealing limiting values close to the theoretical limits for one-photon (0.4) and two-photon (0.57) excitation. The steady-state anisotropy for one-photon excitation decreases in the shorter wavelength region (R6G dye laser, 280 to 315 nm), but the two-photon anisotropy for 560 to 630 nm excitation remains as high as in the long- wavelength region (690 to 770 nm). This result suggests that one- photon absorption is due to two electronic transitions, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm.
Recent experimental results from this laboratory have shown that fluorescence emission can be quenched by the light pulses from cavity-dumped dye lasers, a phenomenon we call `light quenching.' In this overview article we describe some of the possible effects of light quenching on the steady state and time-resolved spectral properties of fluorophores. The extent of light quenching was found to depend on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected and were observed for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Light quenching can decrease or increase the time-zero anisotropy. Our calculations indicate that the anisotropies can increase to unity under selected conditions. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. Quenching with time-delayed light pulses is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. A more complete description of `One and Two-Pulse Theory of Light Quenching,' is presented by Kusba et al. in this same volume. The predicted oscillations have been observed in frequency-domain intensity decay data. Overall, the results suggest a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent modification of the excited state population by the quenching pulse(s), followed by time- or frequency-domain measurements of the resulting emission.
KEYWORDS: Diffusion, Energy transfer, Luminescence, Molecules, Resolution enhancement technologies, Molecular energy transfer, Proteins, Solids, Distance measurement, Fluorescence resonance energy transfer
We describe a method to improve the resolution of donor-to- acceptor distance distributions in molecules which are flexing on the timescale of the fluorescence lifetime. We measured the time- dependent donor decays of two donor (D)-acceptor (A) pairs, where the donor lifetimes were substantially different. The donors were an indole residue (5.7 ns) and a naphthalene residue (24.4 ns). The same dansyl acceptor was used for both D-A pairs. The donor decays are complex due to both a distribution of D-A distances and D-A diffusion. Using the donor decay data for each D-A pair alone, it is difficult to resolve both the distance distribution and the D-to-A diffusion coefficient. However, these values are unambiguously recovered from global analysis of the data from both D-A pairs. Simulations were also used to demonstrate the increased reduction of global analysis with different lifetime donors to obtain distance distribution parameters in the presence of D-A diffusion.
We have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high-repetition rate lasers, a phenomenon we call 'light quenching'. We now describe some of the possible effects of light quenching on the time- resolved intensity and anisotropy decays of fluorophores. We show that light quenching can decrease or increase the time-zero anisotropy, and that the initial anisotropies can increase to unity under selected conditions. Quenching with time-delayed light pulses is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. The increasing available and use of pulsed laser sources of light offers the opportunity for a new class of one- two- or multiple-pulse time- resolved experiments.
Frequency-domain fluorometry was used to investigate the time- dependent intensity decays of N-acetyl-L-tryptophanamide (NATA) when collisionally quenched by acrylamide and iodide in propylene glycol at 20 degree(s)C. The intensity decays of NATA became increasingly heterogeneous in the presence of quenching, and the steady-state Stern-Volmer plots show significant upward curvature. These frequency-domain and steady-state data clearly indicate that the rate constants for quenching of NATA by acrylamide and iodide depend exponentially on the fluorophore- quencher separation distance. The NATA intensity decays were not consistent with the Smoluchowski-Collins-Kimball radiation boundary condition for quenching. The upward curvature of the Stern-Volmer plots provide a sensitive method to determine the characteristic distances for fluorophore-quencher interaction. The rate constant for quenching are discussed based on the mechanism of interaction between tryptophanyl fluorophore and quencher molecules which appear to involve electron transfer for acrylamide and heavy atom effects for iodide. These results suggest that the distance-dependent interaction and the effects of solvent dynamics need to be considered in the interpretations of data for quenching of proteins.
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 intramolecular energy transfer in progressively stretched poly(vinyl alcohol) films were performed. The donor (tryptophan) and acceptor (dansyl) were linked with flexible polymethylene chain. Distance distributions were recovered from frequency-domain measurements of the donor decay. In isotropic PVA (in solution) a wide range of distances were detected (Gaussian full width at half maximum of about 16 angstroms) with an average distance of 13 angstroms. The donor-acceptor distance distribution became progressively more narrow when the PVA films were stretched. Four-fold or more stretching results in a single donor-acceptor distance. The maximal measured donor-acceptor distance of 23 angstroms is in excellent agreement with the computed distance for the fully stretched conformation. The possibility of partial and/or full ordering of polymethylene chains by stretching the PVA films can also be useful in the study of other distance-dependent interactions, such as electron transfer or distance-dependent quenching.
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.
We measure the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10 GHz frequency-domain fluorometer and a specially designed cuvette which allows front face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low intensity fluorescence like in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin is ligand dependent. Fluorescence anisotropy decays of oxy, deoxy, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used the floating initial anisotropy ro is in each case higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.2 occurring in the first two picoseconds.
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 report fluorescence studies with the single trp protein, S. nucelase A, and several of its site-directed mutants. One of these mutants, PA56, which has an alanine at position 56 in place of proline, has a much lower structural stability than the wild type. This is demonstrated by the much lower Tm (30 degrees C) for PA56 than for the wild type (52 degrees C) and by a much lower (urea)1/2 for denaturation of the mutant. Also we show that PA56 can be unfolded by relatively low hydrostatic pressure (~700 bar). The free energy for unfolding of PA56 is found to be only 1.3 kcal/mole (at 20 degrees C) by thermal, urea, quanidine and pressure unfolding. Fluorescence lifetime measurements with wild type nuclease and several of its mutants show non-exponential decay kinetics. The fluorescence decay profiles are similar for the native state of each protein and the decay data at various temperatures generally reveal differences in the Tm for the various mutants. Anisotropy decay data are analyzed in terms of two rotational correlation times, a longer one for overall rotation of the protein and a shorter one for rapid, segemental motion of the trp residue. The mutant PA56 can be easily denatured by temperature, pressure or urea, and anisotropy decay data for these various denatured forms are reported.
We studied energy transfer between tryptophan (donor, D) and dansyl (acceptor, A) separated by three different hexapeptides: hexaglycine, hexaalanine and hexaproline. In each case the donor tryptphanamide was located on the C-terminus, and the dansyl acceptor on the N-terminus amino group. In the absence of acceptor, in propylene glycol at 20°C, tryptophan donors show single exponential fluorescence intensity decays with mean lifetimes about 5.2 ns. In the presence of dansyl, the lifetimes become shorter and intensity decays heterogeneous. However, the extent of heterogeneity is different for each labeled oligopeptide. The strongest heterogeneity was observed for the hexaglycine donor-acceptor pair. Only small deviations from single exponential decay were found for the proline D-A pair. These results suggested a higher degree of conformational heterogeneity for the flexible glycine D-A pair, or composed to the more rigid proline D-A pair. We analyzed the data in terms of Gaussian donor-acceptor distance distributions. The recovered average distances Ray, are 10.7, 15.9 and 23.4 A and full widths at half maximum, hw, are 18.1, 11.0 and 3.9 A for hexaglycine, hexaalanine and hexaproline chain, respectively. The results indicate that flexibility of peptide chain has a strong influence on end-to-end distance distribution. The possible effects of the orientation factor κ2 are discussed, along with the limits on κ2 consistent with the time-dependent anisotropy decays. These distance distributions provide the basis for comparisions and/or refinement of the rotational potential functions of the amino acids.
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.
We used the Hamamatsu model PLP-01 picosecond light pulser as a 413-nm excitation light source for frequency-domain fluorescence measurements. In comparison with sync-pumped/cavity dumped/frequency-doubled dye lasers, the 413-nm PLP-01 shows a longer FWHM (40 ps), a similar pulse repetition rate (up to 10 MHz), much less output power at a fixed wavelength (0.44 mW peak, 220 nW maximum average power), but is less expensive, small-sized, and easy to handle. Using the PLP-01 , we have been able to perform fluorescence measurements up to an upper modulation frequency of about 2000 MHz, and to resolve mixtures of fluorophores exhibiting different lifetimes. During our tests, we observed remarkable and lasting (2 h) time drifts between the optical output and the electrical trigger input or output. At present, work is in progress at Harpamatsu to eliminate these drifts.
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