Fluorescence tomography with simulated data based on the equation of radiative transfer

The quantification of a nonuniform quantum yield or fluorophore absorption distribution is of major interest in molecular imaging of biological tissue. We introduce what is believed to be the first fluorescence image reconstruction algorithm based on the equation of radiative transfer that recovers the spatial distribution of light-emitting fluorophores inside a highly scattering medium from measurements made on the surface of the medium. We obtain images of either the quantum yield or the fluorophore absorption.     

Simultaneous fluorometry and phosphorometry of Langendorff perfused rat heart: ex vivo animal studies

Fluorescence imaging of intrinsic fluorophores of tissue is a powerful method to assess metabolic changes at the cellular and intracellular levels. At the same time, exogenous phosphorescent probes can be used to accurately measure intravascular tissue oxygenation. Heart failure is the leading cause of death in America. A rat heart can potentially model the human heart to study failures or other abnormalities optically. We report simultaneous fluorescence and phosphorescence measurements performed on a rat heart. We have used two different optical systems to acquire fluorescence signals of flavoprotein and nicotinamide adenine dinucleotide--the two intrinsic fluorophores of mitochondria--and the phosphorescence signal of an intravascular oxygen probe to extract intracellular and intravascular metabolism loads, respectively.     

Turbidity-free fluorescence spectroscopy of biological tissue

We present a method based on photon migration of extracting intrinsic fluorescence spectra from turbid media, using concomitantly measured fluorescence and reflectance. Intrinsic fluorescence is defined as fluorescence that is due only to fluorophores, without interference from the absorbers and scatterers that are present. Application to fluorescence spectra taken with tissue phantoms and human mucosal tissues demonstrates excellent agreement in both spectral line shape and intensity between the extracted and the directly measured intrinsic fluorescence spectra.     

Advances in fluorescence diagnosis to track footprints of cancer progression in vivo

Fluorescence spectroscopy and imaging have been widely used for in vivo cancer diagnosis and therapy monitoring in preclinical models, as well as clinical translation. Great attempts have been made to develop novel fluorescence techniques and improve on existing ones, which can now be used in conjunction with newly developed fluorescent probes for specific cancer imaging. In this review, a broad overview of fluorescence techniques is provided, including photodynamic diagnosis, laser confocal endomicroscopy and fluorescence lifetime imaging, coupled with endogenous and exogenous fluorophores. In particular, endogenous fluorophores, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), are highlighted as they are linked to cellular metabolism in precancer growth. The use of near‐infrared dyes, such as indocynanine green (ICG), for imaging deep‐tissue regions is also reviewed. In addition, diagnostic algorithms used for tissue classification and cancer detection will be discussed. Lastly, emerging technologies in fluorescence diagnosis will also be included.     

Light propagation for time-domain fluorescence diffuse optical tomography by convolution using lifetime function

Time-domain light propagation in biological tissue is studied by solving the forward problem for fluorescence diffuse optical tomography using a convolution of the zero-lifetime emission light and the exponential function for a finite lifetime. We firstly formulate the fundamental equations in a time-domain assuming that the fluorescence lifetime is equal to zero, and then the solution including the lifetime is obtained by convolving the emission light and the lifetime function. The model is a two-dimensional (2-D) 10 mm-radius circle with the optical properties simulating biological tissue for the near infrared light, and contains some inclusions with fluorophores. Temporal and spatial profiles of excitation and emission light are calculated and discussed for several models with different inclusions. The results are physically reasonable and will be used for the inverse problem of fluorescence diffuse optical tomography.     

Long-term luminescence of organic dyes in cells of biological tissues

We studied the kinetics of delayed fluorescence and phosphoresce of exogenous fluorophores in cells extracted from tumorous and normal tissues of the mammary gland of mice of the BYRB strain. We revealed the specifics and determined regularities of the kinetics of long-term luminescence of fluorophores in different cells. The possibility of developing of a method for the early fluorescence diagnostics of the pathology of biotissues based on the obtained results is discussed.     

Spatial Localization of Biosensor Fluorescence Signals in Human Skin under the Effect of Eequalization of the Refractive Index of the Surrounding Medium

A priority line of biomedical applications of optics is the development of noninvasive diagnostic methods based on the scanning of fluorescence radiation of biosensors embedded in biological tissue. Their main advantage is a high sensitivity and selectivity with respect to given parameters of tissues and their variations. In this study, we present a method for and results of modeling of excitation and propagation of fluorescence radiation in a multilayer randomly inhomogeneous highly scattering and absorbing medium imitating human skin. The model takes into account the spatially inhomogeneous distribution of skin fluorophores and their photophysical characteristics. Both the spatial distribution of fluorescence of skin tissues and the possibility of localization of a detected fluorescence signal are studied. The spatial distribution of fluorescence centers (fluorophores) in the medium is assumed to closely follow the spatial distribution of collagen fibers of the skin. The equalization of the refractive indices at the air-skin interface is shown to lead to a higher degree of localization of the fluorescence signal detected from a biosensor located in a near-surface skin layer.     

Photoswitching microscopy with standard fluorophores

We introduce far-field subdiffraction-resolution fluorescence imaging based on photoswitching of individual standard fluorophores in air-saturated solution. Here, photoswitching microscopy relies on the light-induced switching of organic fluorophores (ATTO 655 and ATTO 680) into long-lived metastable dark states and spontaneous repopulation of the fluorescent state. In the presence of low concentrations (2–10 mM) of reducing, thiol-containing compounds such as ß-mercaptoethylamine or glutathione, the density of fluorescent molecules can be adjusted to enable multiple localizations of individual fluorophores with an experimental accuracy of ～20 nm. The method requires wide-field illumination with only a single laser beam for readout and photoswitching and provides superresolution fluorescence images of intracellular structures under live cell compatible conditions.     

Fluorescence Spectral Properties of Indocyanine Green on a Roughened Platinum Electrode: Metal-Enhanced Fluorescence

The interactions of fluorophores with noble metal particles can modify their emission spectral properties, a relatively new phenomenon in fluorescence. We subsequently examined indocyanine green (ICG), which is widely used in medical testing and imaging, in close proximity to an electrically roughened platinum electrode. The emission intensity and lifetimes were decreased about 2-fold on the roughened surface as compared to a smooth Pt surface, and the photostability about the same. Platinum does not appear promising for metal enhanced fluorescence, at least for long wavelength fluorophores.     

Fluorescence lifetime system for microscopy and multiwell plate imaging with a blue picosecond diode laser

We report a wide-field fluorescence lifetime imaging (FLIM) system that uses a blue picosecond pulsed diode laser as the excitation source. This represents a significant miniaturization and simplification compared with other time-domain FLIM instruments that should accelerate the development of clinical and real-world applications of FLIM. We have demonstrated this instrument in two configurations: a macroimaging setup applied to multiwell plate assays of chemically and biologically interesting fluorophores and a microscope system that has been applied to imaging of tissue sections. The importance of the adjustable repetition rate of this laser source is discussed with respect to noise reduction and precision in the lifetime determination, illustrating a further significant advantage over conventional mode-locked solid-state lasers.     

Introducing ‘Simple Variable Selection (SVS) Approach’ for Improving the Quantitative Accuracy of Chemometric Assisted Fluorimetric Estimations of Dilute Aqueous Mixtures

Excitation emission matrix fluorescence (EEMF) spectroscopy is a multiparametric fluorescence technique where the fluorescence intensity of a fluorophore is a function of excitation wavelength, emission wavelength and its concentration. The manual analysis of large volume of highly correlated EEMF data sets towards developing a calibration model for quantifying each fluorophores present in multifluorophoric mixtures is a difficult and time-consuming task. Over the years, Partial least square (PLS) algorithm has found its application towards providing swift and efficient analyses of large volumes of highly correlated spectral data sets. The PLS assisted EEMF spectroscopy has been successfully used towards quantifying the fluorophores in multifluorophoric mixtures without involving any pre-separation. However, the accuracy and robustness of developed calibration model can be significantly improved provided PLS analysis is carried out on the analytically relevant EEMF spectral variables. In the present work, a variable selection method baptized as simple variable selection (SVS) approach is introduced that provides a simple and computationally economical means of identifying the useful spectral variables for subsequent PLS analysis. The proposed SVS approach is successfully validated by analyzing the complex EEMF data sets of multifluorophoric mixtures of consisting of multifluorophoric mixtures of biological relevance. The proposed approach is found to provide a simple, swift and efficient means for developing a robust PLS assisted EEMF spectroscopy based calibration model for simultaneous quantification of various fluorophores present in multifluorophoric mixtures.     

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Fluorescein,Quinine Sulfate and Green Fluorescent Protein

It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10^??3 ??10^??5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-photon excitation and lifetime data was obtained in the time-domain using time-correlated single-photon counting.     

Fluorescence polarization imaging for delineating nonmelanoma skin cancers

We present a method for detecting nonmelanoma skin cancers using exogenous fluorescence polarization. We built an automated system that permits exogenous fluorescence polarization imaging. It includes a tunable linearly polarized monochromatic light source and a CCD camera equipped with a rotating linear polarizer and a filter to reject excitation light. Two fluorophores that are retained in tumors, toluidine blue and methylene blue, are employed. We demonstrate that fluorescence polarization imaging can be used for accurate delineation of nonmelanoma cancers. The results suggest that this optical technique may be suitable for real-time noninvasive demarcation of epithelial cancers.     

Enhancing fluorescence detection with a photonic crystal structure in a total-internal-reflection configuration

In contrast to fluorescence enhancement of fluorophores embedded in a photonic crystal structure as previously reported [Appl. Phys. Lett. 75, 3605 (1999)], in this Letter we demonstrate a unique approach to forming an open microcavity using a one-dimensional photonic crystal in a total-internal-reflection geometry. This configuration opens up the possibility for enhancing fluorescence imaging and biosensing. Time-resolved fluorescence detection of fluorophores immobilized on the open cavity has been carried out. Over 20-fold fluorescence enhancement was observed.     

Spectral Properties of Y-Shaped Donor-Acceptor Push-Pull Imidazole-based Fluorophores: Comparison between Solution and Polymer Matrices

The spectral properties of a novel type of Y-shaped fluorophores consisting of an imidazole ring end-capped with two electron-donating N,N-dimethylaminophenyl groups at positions C4 and C5 and one electron-withdrawing cyano group on the imidazole moiety at position C2 were examined. The π-linker separating the 4,5-bis[4-(N,N-dimethylamino)phenyl]-1H-imidazole donor moiety and the cyano group comprises 1,4-phenylene (1), (E)-phenylethenyl (2), (E)-phenylbuta-1,3-dienyl (3), biphenyl (4), (E)-phenylethenylphenyl (5) and phenylethynylphenyl (6) conjugated paths. The absorption and fluorescence spectra were obtained in toluene, dichloromethane, acetonitrile and methanol and in polymer matrices such as polystyrene (PS), poly(methyl methacrylate) (PMMA) and poly(vinylchloride) (PVC). The most intense absorption bands of fluorophores 1-6 were observed within the range of 283 to 330?nm. Less intense but longer-wavelength absorption bands designated as charge-transfer bands were observed at approximately 380-430?nm depending on the medium. The fluorophores exhibited strong fluorescence in the visible region with a Stokes shift of approximately 4300-5800?cm(-1) in non-polar toluene and polystyrene, whereas very low intensity of fluorescence was observed with a Stokes shift in the 6500-7800?cm(-1) region in polar methanol and acetonitrile. The large Stokes shift indicates a large difference in the spatial arrangement of the chromophore in the absorbing and emitting states. A relatively intense fluorescence (quantum yields of 0.12-0.69) was observed only for derivative 1 in all media except methanol. The fluorophores doped in matrices yielded more intense fluorescence compared with the fluorescence in liquid media. The use of solid polymer matrices lowers the probability of forming non-emissive excited states. The fluorescence lifetimes were short (1-4?ns) for all of the fluorophores in solvents and in polymer matrices.     

拟威布尔分布密度函数在荧光寿命成像数据分析中的应用

荧光寿命法成像技术(FLIM)是一种非常有效、功能强大且能用来分析复杂生物组织和细胞分子的成像技术。传统的荧光寿命成像的数据分析,按某些具有不同寿命、离散的单参量指数模型来描述荧光衰减过程。在生物组织这样既复杂又不均匀的样品中,虽然多参量指数模型能提供比单参量指数模型对实验数据更好的拟合效果,但是离散多参量的假定往往是随意的。提出了拟威布尔分布密度函数可能是生物荧光分子团衰减动力过程的真实再现,并且通过计算证明,对于某些生化感兴趣的荧光分子团的多槽基面效价测定样品的数据,相对于单参量指数与多参量指数衰减函数有更好的一致性。同时讨论了将该荧光衰减模型应用于荧光寿命成像的前景。     

Metal-Enhanced Fluorescence from Plastic Substrates

We report the first findings of Metal-Enhanced Fluorescence (MEF) from modified plastic substrates. In the past several years our laboratories have reported the favorable effects of fluorophores in close proximity to silver nanoparticles. These effects include, enhanced fluorescence intensities, (increased detectability), and reduced lifetimes, (enhanced fluorophore photostability). All of these reports have featured silver nanostructures and fluorophores which have been immobilized onto clean glass or quartz surfaces. In this report we show how plastic surfaces can be modified to obtain surface functionality, which in turn allows for silver deposition and therefore metal-enhanced fluorescence of fluorophores positioned above the silver using a protein spacer. Our findings show that plastic substrates are ideal surfaces for metal-enhanced phenomena, producing similar enhancements as compared to clean glass surfaces. Subsequently, we speculate that plastic substrates for MEF will find common place, as compared to the more expensive and less versatile traditional silica based supports.     

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.     

Experimental three-dimensional fluorescence reconstruction of diffuse media by use of a normalized Born approximation

We present a normalized Born expansion that facilitates fluorescence reconstructions in turbid, tissuelike media. The algorithm can be particularly useful for tissue investigations of fluorochrome distributionin vivo, since it does not require absolute photon-field measurements or measurements before contrast-agent administration. This unique advantage can be achieved only in fluorescence mode. We used this algorithm to three-dimensionally image and quantify an indocyanine fluorochrome phantom, using a novel fluorescence tomographic imager developed for animals.     

Fluorescence lifetime imaging with a low-repetition-rate passively mode-locked diode-pumped Nd:YVO4 oscillator

We report a wide-field fluorescence lifetime imaging microscope based on a low-repetition-rate (3.7-MHz) passively mode-locked diode-pumped laser source. This inexpensive and compact laser source operating in the visible and UV range can excite a wide range of fluorophores of biological interest. We demonstrate that the power of this laser source is highly sufficient for studying biological systems with low quantum yields (autofluorescence of tissues and stained living cells). The maximum measurable lifetime is also strongly increased with this laser source, as fluorescence intensity measurement can occur 250 ns after the excitation pulse.