Vertex/propagator model for least-scattered photons traversing a turbid medium

The least-scattered photons that arrive at a detector through highly scattering tissues have the potential to image internal structures, functions, and status with high imaging resolution. In contrast, optical diffusing tomography is based on the use of the late-arriving photons, which have been diffusely scattered, leading to very low imaging resolution. A good model of the early-arriving photons, i.e., the least-scattered photons, may have a significant effect on the development of imaging algorithms and a further understanding of imaging mechanisms within current high-resolution optical-imaging techniques. We describe a vertex/propagator approach that attempts to find the probabilities for least-scattered photons traversing a scattering medium, based on analytical expressions for photon histories. The basic mathematical derivations for the model are outlined, and the results are discussed and found to be in very good agreement with those from the Monte Carlo simulations.     

Imaging diffusion in living cells using time-correlated single-photon counting

Current efforts to monitor the diffusion of proteins in living cells are based on either fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching, or image correlation spectroscopy. However, these methods cannot generate a map of diffusion times. Here, we introduce a new method termed diffusion imaging microscopy that combines scanning confocal microscopy, time-correlated single-photon counting, and FCS and thus allows us to measure spatially resolved diffusion times. In our approach, we record scan images with time-resolved photon streams within each individual pixel. By extending the pixel dwell time to 25-100 ms, a software correlation of individual photons within each pixel yields the average diffusion time. Additionally, information on fluorescence intensity (number of photons) and fluorescence lifetime is available and can be used to sort fluorescence photons and to discriminate from autofluorescence. We evaluated our method by measuring diffusion times of dT20-TMR in solutions of different viscosity. We further demonstrate the applicability of the method to living cells and recorded a diffusion map of a living 3T3 mouse fibroblast incubated with dT20-ATTO488.     

Comparative study of X-ray fluorescence signal collection by collimators of two types

The response signal collection functions in a confocal setup of the X-ray fluorescence (XRF) tomography have been modeled for a polycapillary collimator and a multichannel collimator fabricated by means of microelectronic technology (MT). The dimensions of the effective collection region for the two types of collimators are compared. For the MT-fabricated device, a fraction of X-ray photons transmitted due to the total external reflection from channel walls has been evaluated. It is shown that, for a proper choice of parameters, the contribution of these photons to the collection function is negligibly small.     

Three-dimensional imaging of objects embedded in turbid media with fluorescence and Raman spectroscopy

We present a single-ended technique for three-dimensional imaging of objects embedded in a turbid medium by the use of time-resolved fluorescence emission or Raman scattering. The technique uses the earliest arriving photons, which we show are not sensitive to the relatively long fluorescence lifetime, and thus can be used to extract the desired spatial information accurately, even at a distance equivalent to 100 mean free paths. The results also demonstrate the feasibility and the potential of one's combining time-resolved optical tomography with fluorescence or Raman spectroscopy to localize and identify the embedded objects. This technique may be valuable for the diagnosis of disease in highly scattering human tissue because it can provide spatial and biochemical information about the composition of embedded lesions.     

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.     

Robust inference of baseline optical properties of the human head with three-dimensional segmentation from magnetic resonance imaging

We model the capability of a small (6-optode) time-resolved diffuse optical tomography (DOT) system to infer baseline absorption and reduced scattering coefficients of the tissues of the human head (scalp, skull, and brain). Our heterogeneous three-dimensional diffusion forward model uses tissue geometry from segmented magnetic resonance (MR) data. Handling the inverse problem by use of Bayesian inference and introducing a realistic noise model, we predict coefficient error bars in terms of detected photon number and assumed model error. We demonstrate the large improvement that a MR-segmented model can provide: 2-10% error in brain coefficients (for 2 x 10(6) photons, 5% model error). We sample from the exact posterior and show robustness to numerical model error. This opens up the possibility of simultaneous DOT and MR for quantitative cortically constrained functional neuroimaging.     

一种潜在的激光制冷介质-罗丹明B掺杂PMMA

报道了罗丹明B掺杂PMMA材料制作过程及材料吸收和荧光光谱实验。吸收光谱表明，PMMA样品从紫外到近红外范围存在较小的吸收。罗丹明B／PMMA样品吸收光谱主要反映罗丹明B的吸收特征，其吸收峰中心波长位于550nm。荧光光谱显示，以630nm激发，反斯托克斯荧光峰位于595nm，能量差为0．11eV。该材料辐射反斯托克斯荧光，可用于激光制冷领域的研究。     

Effects of background fluorescence in fluorescence molecular tomography

Recent advances in optical imaging systems and systemically administered fluorescent probes have significantly improved the ways by which we can visualize proteomics in vivo. A key component in the design of fluorescent probes is a favorable biodistribution, i.e., localization only in the targeted diseased tissue, in order to achieve high contrast and good detection characteristics. In practice, however, there is always some level of background fluorescence present that could result in distorted or obscured visualization and quantification of measured signals. In this study we observe the effects of background fluorescence in tomographic imaging. We demonstrate that increasing levels of background fluorescence result in artifacts when using a linear perturbation algorithm, along with a significant loss of image fidelity and quantification accuracy. To correct for effects of background fluorescence, we have applied cubic polynomial fits to bulk raw measurements obtained from spatially homogeneous and heterogeneous phantoms. We show that subtraction of the average fluorescence response from the raw data before reconstruction can improve image quality and quantification accuracy as shown in relatively homogeneous or heterogeneous phantoms. Subtraction methods thus appear to be a promising route for adaptively correcting nonspecific background fluorochrome distribution.     

Noncontact fluorescence diffuse optical tomography of heterogeneous media

Fluorescence-enhanced diffuse optical tomography is expected to be useful to the collection of functional information from small animal models. This technique is currently limited by the extent of tissue heterogeneity and management of the shape of the animals. We propose an approach based on the reconstruction of object heterogeneity, which provides an original solution to the two problems. Three evaluation campaigns are described: the first two were performed on phantoms designed to test the reconstructions in highly heterogeneous media and noncontact geometries; the third was conducted on mice with lung tumors to test fluorescence yield reconstruction feasibility in vivo.     

Generalized diffusion model in optical tomography with clear layers

We introduce a generalized diffusion equation that models the propagation of photons in highly scattering domains with thin nonscattering clear layers. Classical diffusion models break down in the presence of clear layers. The model that we propose accurately accounts for the clear-layer effects and has a computational cost comparable to that of classical diffusion. It is based on modeling the propagation in the clear layer as a local tangential diffusion process. It can be justified mathematically in the limit of small mean free paths and is shown numerically to be very accurate in two- and three-dimensional idealized cases. We believe that this model can be used as an accurate forward model in optical tomography.     

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.     

Mathematical model of fluorescence endoscopic image formation

We present a mathematical model that describes the spatial distribution of photons in fluorescence endoscopic images, resulting in expressions for image signal-to-noise ratio and resolution. This model was applied to quantitative analysis of fluorescence images collected from human colonic mucosa with a fiber-optic and an electronic endoscope. It provides a tool for the design of fluorescence endoscopic imaging systems and for extraction of quantitative information about image features. The results apply generally to endoscopic imaging of remote structures in biological and industrial settings, in which light of weak intensity such as fluorescence as well as reflected white light is used.     

Epifluorescence collection in two-photon microscopy

We present a simple model to describe epifluorescence collection in two-photon microscopy when one images in a turbid slab with an objective. Bulk and surface scattering determine the spatial and angular distributions of the outgoing fluorescence photons at the slab surface, and geometrical optics determines how efficiently the photons are collected. The collection optics are parameterized by the objective's numerical aperture and working distance and by an effective collection field of view. We identify the roles of each of these parameters and provide simple rules of thumb for the optimization of the epifluorescence collection efficiency. Analytical results are corroborated by Monte Carlo simulation.     

Fluorescence lifetime imaging with near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.     

Distribution of the paths of early-arriving photons traversing a turbid medium

We describe experiments to measure the spatial and the temporal distribution of photons traversing a turbid medium in the early-arriving regime in which the photons are multiply scattered but are not completely randomized. The photon paths are resolved temporally by a streak camera and spatially by an adjustable absorbing screen with a small aperture. The results are compared with predictions of a theory based on path integrals (PIs) and with the standard diffusion approximation. The PI theory agrees with the data for both long and short times of flight; this agreement is in contrast to the diffusion approximation, which fails for short times. An alternative PI calculation, based on the use of an effective Lagrangian, also agrees with the experiments. PI theory succeeds because it preserves causality. The implications for optical tomography are discussed.     

Photon path distribution in inhomogeneous turbid media: theoretical analysis and a method of calculation

The photon path distribution (PPD) is a measure that I have developed to express optical responses in inhomogeneous turbid media in the time and frequency domains. The PPD is defined by local photon pathlengths of possible photons having total zigzag pathlengths I between the points of light input and detection. Such a distribution is independent of absorption and is uniquely determined for the medium under quantification. I show that the PPD is derived through the local photon count of the possible photons arising from an optical impulse incident on an imaginary medium having the same optical properties as the medium under quantification, except for the absence of absorption. The formulas derived can be used to calculate the PPD simultaneously with, for example, the numerical calculation of a diffusion equation.     

Search for ultra-high energy photons with the Pierre Auger Observatory

The Pierre Auger Observatory [1] has an unprecedented sensitivity to photons at energies above 10¹⁸ eV. Particularly the combination of ground array and fluorescence detection techniques offers a unique power to discriminate the primary particles based on different observables. Implications of photon searches extend from astrophysics to fundamental and particle physics. Current results and future prospects are reported.     

Label-free detection of single protein molecules using deep UV fluorescence lifetime microscopy

We present the detection of single beta-galactosidase molecules from Escherichia coli (Ecbeta Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecbeta Gal protein as well as fluorescence correlation spectroscopy of Ecbeta Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence.     

Heterogeneity assessment in individual CaCO3-CaSO4 particles using ultrathin window electron probe X-ray microanalysis

In our previous studies, it has been demonstrated that both the excitation interactions between electrons and the atoms of the matrix and the matrix and geometric effects of electron-induced X-ray signals can be described by Monte Carlo simulation for low-Z elements, such as carbon, nitrogen, and oxygen, in individual atmospheric microparticles. In addition, by the application of a quantification method, which employs Monte Carlo simulation combined with successive approximations, at least semi-quantitative specification of the chemical compositions could be done. This has enlarged the scope of electron probe X-ray microanalysis (EPMA) for the single particle analysis of atmospheric environmental aerosol particles. In this work, we demonstrate that the heterogeneity of individual particles, even of micrometer size, can be characterized by the application of EPMA. X-ray photons obtained with different primary electron beam energies carry information on the chemical compositions for different regions in the particles. Artificially generated heterogeneous CaCO3-CaSO4 individual particles were measured at different accelerating voltages, and it was found that the Monte Carlo calculation is a powerful technique to extract the information on the heterogeneity of the particles that is contained in the measured X-ray data. Our approach can even estimate the thickness of the surface CaSO4 species by the application of the Monte Carlo calculation. A preliminary result for carbon-coated glass particles is also presented. The complexity involved in the analysis of real world particles is briefly mentioned with a result for heterogeneous SiO2 particle.