Frequency-domain fluorescent diffusion tomography: a finite-element-based algorithm and simulations

We present a finite-element-based algorithm for reconstruction of fluorescence lifetime and yield in turbid media, using frequency-domain data. The algorithm is based on a set of coupled diffusion equations that describe the propagation of both excitation and fluorescent emission light in multiply scattering media. Centered on Newton's iterative method, we implemented our algorithm by using a synthesized scheme of Marquardt and Tikhonov regularizations. A low-pass spatial filter is also incorporated into the algorithm for enhancing image reconstruction. Simulation studies using both noise-free and noisy data have been performed with the nonzero photon density boundary conditions. Our results suggest that quantitative images can be produced in terms of fluorescent lifetime and yield values and location, size, and shape of heterogeneities within a circular background region.     

Fluorescent heterogeneities in turbid media: limits for detection, characterization, and comparison with absorption

The fundamental limits for detection and characterization of fluorescent (phosphorescent) inhomogeneities embedded in tissuelike highly scattering turbid media are investigated. The absorption and fluorescence contrast introduced by exogenous fluorophores are also compared. Both analyses are based on practical signal-to-noise ratio considerations. For an object with fivefold fluorophore concentration and lifetime contrast with respect to the background tissue, we find the smallest detectable fluorescent object at 3-cm depth in tissuelike turbid media to be ~0.25 cm in radius, whereas the smallest characterizable object size is ~0.75 cm in radius, given a model with 1% amplitude and 0.5 degrees phase noise. We also find that, for fluorescence extinction coefficients epsilon 相似文献   

Investigation of the Co‐Dependence of Morphology and Fluorescence Lifetime in a Metal‐Organic Framework

Porous materials, due to their large surface‐to‐volume ratio, are important for a broad range of applications and are the subject of intense research. Most studies investigate the bulk properties of these materials, which are not sensitive to the effect of heterogeneities within the sample. Herein, a new strategy based on correlative fluorescence lifetime imaging and scanning electron microscopy is presented that allows the detection and localization of those heterogeneities, and connects them to morphological and structural features of the material. By applying this method to a dye‐modified metal‐organic framework (MOF), two independent fluorescence quenching mechanisms in the MOF scaffold are identified and quantified. The first mechanism is based on quenching via amino groups, while the second mechanism is influenced by morphology. Furthermore, a similar correlation between the inherent luminescence lifetime and the morphology of the unmodified MOF structure is demonstrated.     

Imaging of fluorescent yield and lifetime from multiply scattered light reemitted from random media

The feasibility of employing fluorescent contrast agents to perform optical imaging in tissues and other scattering media has been examined through computational studies. Fluorescence lifetime and yield can give crucial information about local metabolite concentrations or environmental conditions within tissues. This information can be employed toward disease detection, diagnosis, and treatment if noninvasively quantitated from reemitted optical signals. However, the problem of inverse image reconstruction of fluorescence yield and lifetime is complicated because of the highly scattering nature of the tissue. Here a light propagation model employing the diffusion equation is used to account for the scattering of both the excitation and fluorescent light. Simulated measurements of frequency-domain parameters of fluorescent modulated ac amplitude and phase lag are used as inputs to an inverse image-reconstruction algorithm, which employs the diffusion model to predict frequency-domain measurements resulting from a modulated input at the phantom periphery. In the inverse image-reconstruction algorithm, a Newton-Raphson technique combined with a Marquardt algorithm is employed to converge on the fluorescent properties within the medium. The successful reconstruction of both the fluorescence yield and lifetime in the case of a heterogeneous fluorophore distribution within a scattering medium has been demonstrated without a priori information or without the necessity of obtaining absence images.     

Fluorescent diffuse photon density waves in homogeneous and heterogeneous turbid media: analytic solutions and applications

We present analytic solutions for fluorescent diffuse photon density waves originating from fluorophores distributed in thick turbid media. Solutions are derived for a homogeneous turbid medium containing a uniform distribution of fluorophores and for a system that is homogeneous except for the presence of a single spherical inhomogeneity Generally the inhomogeneity has fluorophore concentration, and lifetime and optical properties that differ from those of the background. The analytic solutions are verified by numerical calculations and are used to determine the fluorophore lifetime and concentration changes required for the accurate detection of inhomogeneities in biologically relevant systems. The relative sensitivities of absorption and fluorescence methods are compared.     

Clinical imaging fluorescence apparatus for the endoscopic photodetection of early cancers by use of Photofrin II

A fluorescence imaging device applied to the detection of early cancer is described. The apparatus is based on the imaging of laser-induced fluorescence of a dye that localizes in a tumor with a higher concentration than in the surrounding normal tissue after iv injection. Tests carried out in the upper aerodigestive tract, the tracheobronchial tree, and the esophagus with Photofrin II (1 mg/kg of body weight) as the fluorescent agent are reported as examples. The fluorescence is induced by violet (410-nm) light from a continuous-wave (cw) krypton-ion laser. The fluorescence contrast between tumor and surrounding tissue is enhanced by real-time image processing. This is done by the simultaneous recording of the fluorescence image in two spectral domains (470-600 and 600-720 nm), after which these two images are digitized and manipulated with a mathematical operator (look-up table) at video frequency. Among the 7 photodetections performed in the tracheobronchial tree, 6 were successful, whereas it was the case for only 5 of the 15 lesions investigated in squamous mucosa (upper aerodigestive tract and esophagus). The sources of false positives and false negatives are evaluated in terms of the fluorescent dye, tissue optical properties, and illumination optics.     

Capillary waveguide optrodes: an approach to optical sensing in medical diagnostics

Glass capillaries with a chemically sensitive coating on the inner surface are used as optical sensors for medical diagnostics. A capillary simultaneously serves as a sample compartment, a sensor element, and an inhomogeneous optical waveguide. Various detection schemes based on absorption, fluorescence intensity, or fluorescence lifetime are described. In absorption-based capillary waveguide optrodes the absorption in the sensor layer is analyte dependent; hence light transmission along the inhomogeneous waveguiding structure formed by the capillary wall and the sensing layer is a function of the analyte concentration. Similarly, in fluorescence-based capillary optrodes the fluorescence intensity or the fluorescence lifetime of an indicator dye fixed in the sensing layer is analyte dependent; thus the specific property of fluorescent light excited in the sensing layer and thereafter guided along the inhomogeneous waveguiding structure is a function of the analyte concentration. Both schemes are experimentally demonstrated, one with carbon dioxide as the analyte and the other one with oxygen. The device combines optical sensors with the standard glass capillaries usually applied to gather blood drops from fingertips, to yield a versatile diagnostic instrument, integrating the sample compartment, the optical sensor, and the light-collecting optics into a single piece. This ensures enhanced sensor performance as well as improved handling compared with other sensors.     

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.     

Application of size exclusion electrochromatography to the microanalytical determination of the molecular mass distribution of celluloses from objects of cultural and historical value

The molecular mass distributions (MMD) of celluloses from paper and other sources were determined by size exclusion electrochromatography (SEEC). Prior to the separation the celluloses were chemically modified with phenyl isocyanate into their tricarbanilate derivatives (CTC). Sensitive UV detection of the CTC could be performed at a wavelength of 210 nm. The solvent used for separation was acetone. With this solvent, a high electroosmotic flow could be generated in columns packed with bare silica particles. With a column packed with particles with a nominal pore size of 30 nm, use of a mobile phase with a salt concentration of 0.1 mM was found to be optimal with respect to mass selectivity and efficiency. The workable mass range under these conditions was from 2 kDa to at least 500 kDa for (native) celluloses. The SEEC method was compared to classical pressure-driven size exclusion chromatography (PD-SEC). It is shown that the two methods give comparable results for the MMD of celluloses, while SEEC has important advantages in terms of speed and sample consumption. With SEEC, the analysis time was less than 20 min. The method was applied for the study of cellulose degradation during (artificial) aging of paper samples. A clear reduction of the average molecular mass of cellulose during aging was observed. With SEEC, the required sample amount is strongly reduced compared to classical PD-SEC. With a single paper fiber (after derivatization), multiple analyses could be carried out. It is argued that this is not only important for the analysis of unique objects but that it also allows the detection of MMD heterogeneities on a microscale. The strong reduction in sample size may be relevant when local heterogeneities in other types of polymer samples are studied.     

Ratiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c' and the detection of extra- and intracellular macrophage nitric oxide.

Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c', a hemoprotein known to bind nitric oxide selectively. The cytochrome c' is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c' attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (< or = 0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 microM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c' in solution and have a limit of detection of 30 microM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 +/- 90 microM for activated macrophages, while an average intracellular concentration of 160 +/- 10 microM was determined from the lifetime-based measurements of dye-labeled cytochrome c' in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.     

全反射法折射率测量中的多光源光学系统设计

介绍了一种用于全反射法折射率测量的光学系统.当采用一个光源时,折射率测量的精度和量程受到接收面积的限制,采用多光源的光学系统,通过把量程分段,切换不同的光源来选择合适的量程,能在不改变接收端光电传感器的条件下,提高测量精度,扩大量程.本文根据折反射定律,通过分析光路图,给出了此光学系统的结构参数.理论计算和实验结果表明,当采用三个光源时,测量精度或量程比单光源系统提高到三倍左右.     

Nanoparticle-induced fluorescence lifetime modification as nanoscopic ruler: demonstration at the single molecule level

We combine interferometric detection of single gold nanoparticles, single molecule microscopy, and fluorescence lifetime measurement to study the modification of the fluorescence decay rate of an emitter close to a nanoparticle. In our experiment, gold particles with a diameter of 15 nm were attached to single dye molecules via double-stranded DNA of different lengths. Nanoparticle-induced lifetime modification (NPILM) has promise in serving as a nanoscopic ruler for the distance range well beyond 10 nm, which is the upper limit of fluorescence resonant energy transfer (FRET). Furthermore, the simultaneous detection of single nanoparticles and fluorescent molecules presented in this work provides new opportunities for single molecule biophysical studies.     

Characterization of time-resolved fluorescence response measurements for distributed optical-fiber sensing

A distributed optical-fiber sensing system based on pulsed excitation and time-gated photon counting has been used to locate a fluorescent region along the fiber. The complex Alq3 and the infrared dye IR-125 were examined with 405 and 780 nm excitation, respectively. A model to characterize the response of the distributed fluorescence sensor to a Gaussian input pulse was developed and tested. Analysis of the Alq3 fluorescent response confirmed the validity of the model and enabled the fluorescence lifetime to be determined. The intrinsic lifetime obtained (18.2±0.9 ns) is in good agreement with published data. The decay rate was found to be proportional to concentration, which is indicative of collisional deactivation. The model allows the spatial resolution of a distributed sensing system to be improved for fluorophores with lifetimes that are longer than the resolution of the sensing system.     

Sensitive and selective detection of adenine using fluorescent ZnS nanoparticles

We have used fluorescent ZnS nanoparticles as a probe for the determination of adenine. A typical 2 × 10(-7) M concentration of adenine quenches 39.3% of the ZnS fluorescence. The decrease in ZnS fluorescence as a function of adenine concentration was found to be linear in the concentration range 5 × 10(-9)-2 × 10(-7) M. The limit of detection (LOD) of adenine by this method is 3 nM. Among the DNA bases, only adenine quenched the fluorescence of ZnS nanoparticles in the submicromolar concentration range, thus adding selectivity to the method. The amino group of adenine was important in determining the quenching efficiency. Steady-state fluorescence experiments suggest that one molecule of adenine is sufficient to quench the emission arising from a cluster of ZnS consisting of about 20 molecules. Time-resolved fluorescence measurements indicate that the adenine molecules block the sites on the surface of ZnS responsible for emission with the longest lifetime component. This method may be applied for the determination of adenine in biological samples since the measurements have been carried out at pH 7.     

Long-path measurement of atmospheric NO2 with an obstruction flashlight and a charge-coupled-device spectrometer

A novel method of differential optical absorption spectroscopy (DOAS) is proposed and demonstrated to monitor the concentration of atmospheric pollutant gas. In contrast to conventional DOAS measurements with continuous light sources, the present method relies on white flashlights such as aviation obstruction lights that are generally installed on tall constructions. A simple detection system is devised by means of a telescope and a compact CCD spectrometer. A path length of 5.5 km allows us to measure atmospheric NO2 concentration with a detection limit of approximately 1 part per billion. We also discuss the possibility of deriving the aerosol optical thickness through the horizontal atmosphere from this pulsed DOAS measurement.     

Multiple-source optical diffusion approximation for a multilayer scattering medium

A method for improving the accuracy of the optical diffusion theory for a multilayer scattering medium is presented. An infinitesimally narrow incident light beam is replaced by multiple isotropic point sources of different strengths that are placed in the scattering medium along the incident beam. The multiple sources are then used to develop a multilayer diffusion theory. Diffuse reflectance is then computed using the multilayer diffusion theory and compared with accurate data computed by the Monte Carlo method. This multisource method is found to be significantly more accurate than the previous single-source method.     

UV fluorescence lifetime imaging microscopy: a label-free method for detection and quantification of protein interactions

Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm.To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the picomolar concentration range. The obtained affinity constant, Ka, of (1.4 +/- 0.6) x 10(9) M(-1), represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays.     

Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate

We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species: Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.     

Effects of acoustic heterogeneity in breast thermoacoustic tomography

The effects of wavefront distortions induced by acoustic heterogeneities in breast thermoacoustic tomography (TAT) are studied. Amplitude distortions are shown to be insignificant for different scales of acoustic heterogeneities. For wavelength-scale, or smaller, heterogeneities, amplitude distortion of the wavefront is minor as a result of diffraction when the detectors are placed in the far field of the heterogeneities. For larger-scale heterogeneities at the parenchyma wall, by using a ray approach (geometric optics), we show that no refraction-induced multipath interference occurs and, consequently, that no severe amplitude distortion, such as is found in ultrasound tomography, exists. Next, we consider the effects of phase distortions (errors in time-of-flight) in our numerical studies. The numerical results on the spreads of point sources and boundaries caused by the phase distortions are in good agreement with the proposed formula. After that, we demonstrate that the blurring of images can be compensated for by using the distribution of acoustic velocity in the tissues in the reconstructions. The effects of the errors in the acoustical velocities on this compensation also are investigated. An approach to implement the compensation using only TAT data is proposed. Lastly, the differences in the effects of acoustic heterogeneity and the generation of speckles in breast TAT and breast ultrasound imaging are discussed.     

On-the-fly fluorescence lifetime detection of humic substances in capillary electrophoresis

On-the-fly fluorescence lifetime detection was investigated as a tool for studying humic substances in capillary zone electrophoresis (CZE). Humic substances are complex, heterogeneous mixtures of natural products that tend to migrate in a single, broad CZE peak. The intrinsic fluorescence lifetime of five humic substances from the International Humic Substances Society (IHSS) was monitored using excitation at 488 or 364 nm to produce intensity-lifetime electropherograms for each of the substances. Each frequency-domain lifetime measurement, collected at subsecond intervals during the CZE run, contains the equivalent of a complete decay profile. Lifetime analysis of each decay profile was used to construct a lifetime-resolved electropherogram for each lifetime component, from which the variation in relative intensity contributions of each lifetime across the broad CZE peak could be determined. Absorption spectra, fluorescence excitation-emission spectra, and lifetime profiles of batch solutions of the samples were determined as well. It was found that, whereas absorption and fluorescence spectral characteristics tended to discriminate between humic acids and fulvic acids, the batch solution lifetime profiles discriminated instead between samples from different sources, regardless of fraction. On-the-fly lifetime detection provided a more detailed view of the fluorescence decay of the samples, including greater resolution of lifetimes for two of the fulvic acids and greater discrimination among samples based on lifetime profiles across the CZE peaks.