Time-resolved fluorescence intensity issued from a heterogeneous slab: Sensitivity characterization

Optical imaging using fluorescent contrast agents has become an interesting tool to differentiate diseased lesions from normal tissue. However, several sensitivity characterizations may strongly influence the time-dependent fluorescence measurements. Herein, we present a numerical model based on the finite element method that allows the simulation of time-resolved reflectance and transmittance signals from heterogeneous media mimicking breast tissues with an embedded fluorescent object (tumor). The influence, on the computed signals, of several tumor depths, as well as various fluorophore concentrations and several fluorescent markers targeting are analyzed. The results show the possibility of uncoupling location depth from the shape of the target. Therefore, the analysis of the time to reach half the maximum intensity is validated as a good localization scheme. Then, the transmitted data show that the maximal detected intensity at the bottom of the medium is very sensitive to the dye concentration but not to the tumor shape. Moreover, the strong competition between concentration determination and fluorophore distribution is presented. These results will lead to a better detection and localization of tumors.     

Fluorescence lifetime tomography of live cells expressing enhanced green fluorescent protein embedded in a scattering medium exhibiting background autofluorescence

We present a novel fluorescence lifetime tomography system applied to a highly scattering autofluorescent phantom containing live cells expressing the fluorophore enhanced green fluorescent protein (EGFP). The fluorescence signal was excited using a fiber-laser-pumped supercontinuum source and detected using wide-field time gating imaging. To facilitate rapid 3D reconstruction of the fluorescence lifetime distribution, the time-resolved data were Fourier-transformed in time to give complex functions that formed a data set for the Fourier domain reconstruction. Initially the presence of an unspecified background autofluorescence signal impeded reconstruction of the lifetime distribution, but we show that this problem can be addressed using a simple iterative technique.     

Ultrafast fluorescence spectroscopy for axial resolution of flurorophore distributions

A new method for determining the fluorophore distribution along the propagation axis of an ultrashort optical pulse is presented. The axial resolution is obtained by temporal gating of the backward emitted fluorescence via optical parametric amplification, and we demonstrated a resolution in the order of a few 100 μm. With this approach, sampling of the fluorophore concentration of thin layers without using optics with a large numerical aperture will be possible, such as investigating the human retina via time-resolved fluorescence measurements. Additionally, we verified the gain is orders of magnitude higher for coherent seeding, making optical parametric gating very interesting for discriminating between coherently and incoherently scattered light for other multimodal imaging applications.     

Do fluorescence decays remitted from tissues accurately reflect intrinsic fluorophore lifetimes?

Fluorescence spectroscopy and imaging methods, including fluorescence lifetime sensing, are being developed for noninvasive tissue diagnostics. The purpose of this study was to identify and quantify those factors affecting the accurate recovery of fluorophore lifetimes from inhomogeneous tissues in vivo. A Monte Carlo code was developed to numerically simulate time-resolved fluorescence measurements on layered epithelial tissues. Simulations were run with experimental parameters matching previously reported clinical studies in the gastrointestinal tract. The results demonstrate that variations in fluorescence decay time as large as those detected clinically between normal and premalignant tissues (approximately 2 ns) could be simulated by variations in tissue morphology or biochemistry, even when intrinsic fluorophore lifetimes were held constant.     

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.     

Computer simulation of time-resolved optical imaging of objects hidden in turbid media

We review research on time-resolved optical imaging of objects hidden in strongly scattering media, with emphasis on the application to breast cancer detection. A method is presented to simulate the propagation of light in turbid media. Based on a numerical algorithm to solve the time-dependent diffusion equation, the method takes into account spatial variations of the reduced scattering and absorption factors of the medium due to the presence of objects as well as random fluctuations of these factors. It is shown that the simulation method reproduces, without fitting, experimental results on tissue-like phantoms. Using experimental and simulation results, an assessment is made of the reliability for extracting the reduced scattering and absorption coefficients of the medium from time-resolved reflection and transillumination data. The simulation technique is employed to study the conditions for locating mm-sized objects immersed in a turbid medium, by direct, time-resolved imaging. We discuss a simple method to enhance the imaging power of the time-resolved technique. The mathematical justification of the method, as well as some applications to simple problems, is given. The simulation technique is employed to demonstrate the effectiveness of the data processing technique. Results of time-resolved reflection experiments and simulations are presented, showing that the use of the latter allow us to locate 1 mm diameter objects under conditions which would prevent detection otherwise. Our results demonstrate that the combination of simulation and the appropriate processing of the diffusive part of the time-resolved reflected or transmitted light intensity may substantially increase the potential of the time-resolved near-infrared diffusive light imaging technique as a diagnostic tool for breast cancer detection.     

New Perspectives of Fluorescence Correlation Spectroscopy

The principle of fluorescence correlation spectroscopy is outlined. The technique has been applied to a mutant of the well-known green fluorescent protein. A comparative study has been made with time-resolved fluorescence anisotropy. The latter experiment shows that the fluorophore is rigidly bound inside the protein matrix follows the rotation of the whole protein and does not show any fast restricted motion. It is evident from fluorescence correlation spectroscopy that some excited-state reaction plays a role, since the autocorrelation traces show a significant effect on the incident laser power. Other potential applications of fluorescence correlation spectroscopy are presented as taken from very recent publications.     

Time-resolved fluorescence anisotropy imaging applied to live cells

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.     

Simple time-domain optical method for estimating the depth and concentration of a fluorescent inclusion in a turbid medium

A simple time-domain optical method for estimating the depth and concentration of fluorescent inclusions in turbid media is described. We demonstrate direct depth estimation of a localized fluorescent object from the temporal position of the temporal point-spread function maximum. The depth estimation permits recovery of the fluorophore concentration, both of which are essential quantities for optical molecular imaging studies. Since the maximum is independent of the fluorophore concentration, excitation laser power, detector gain, and other system-dependent factors, this method ensures a robust and efficient approach.     

Time-resolved imaging of fluorescent inclusions in optically turbid medium — phantom study

We present results of application of a time-resolved optical system for imaging of fluorescence excited in an inclusion containing indocyanine green (ICG), and located in optically turbid medium. The developed imaging system enabled simultaneous acquisition of fluorescence and diffusive reflectance. Eight independent time-resolved measurement channels based on time-correlated single photon counting technique were applied. In four of these channels, used for the fluorescence detection, sets of filters were applied in order to block the excitation light. Fast optomechanical switches allowed us to illuminate sequentially nine different spots on the surface of the studied object and finally 4×4 pixels maps at excitation and emission wavelengths were obtained. A liquid phantom used in this study consists of the fish tank filed with a solution ofmilk and water with black ink added to obtain optical properties in the range of the optical properties typical for the living tissue. A gel ball of a diameter of 5 mm with precisely controlled concentration of ICG was immersed in the liquid. The measurements were performed for inclusion located at different depths and for various ICG concentrations in the gel ball and in the surrounding liquid. The recorded distributions of times of arrival (DTA) of fluorescence photons and times of flight (DTOF) of diffusely reflected photons were analyzed by calculation of their statistical moments. We observed specific changes in moments of the measured DTAs as a function of depth of immersion of the fluorescent inclusion in the medium. We noted also that the changes of moments depend significantly on concentration of the dye in the fluorescence inclusion as well as in the surrounding liquid.     

Full-field time-resolved fluorescence tomography of small animals

In this experimental investigation, we explore the feasibility of using wide-field illumination for time-resolved fluorescence molecular tomography. The performance of wide-field patterns with a time-resolved imaging platform is investigated in vitro and in a small animal model. A Monte Carlo-based forward model is employed to reconstruct fluorescence yield based on time-gated datasets. An improvement in resolution and quantification when using the time-gate data type compared to the commonly used cw data type is demonstrated in vitro. Furthermore, the feasibility of wide-field strategies for fluorescence preclinical applications is established by an accurate localization of a fluorescent inclusion implanted in the chest cavity of a murine model.     

Fluorescence Origin of 6,P-toluidinyl-naphthalene-2-sulfonate (TNS) Bound to Proteins

6,P-toluidinylnaphthalene-2-sulfonate (TNS) is a highly fluorescent molecule when dissolved in a low polarity medium or when bound to proteins. The aim of the present work is to explain origin of this fluorescence, to find out how the medium (solvent, protein matrix) affects fluorescence observables such as lifetimes and spectra and finally to put into evidence possible relation that exists between these observables and fluorophore structure. To achieve our goal we performed studies on TNS dissolved in ethanol, at high concentrations in water (aggregated form) and bound to proteins. Our experiments allowed us to find out that TNS in the three environments has different structures. Presence of three lifetimes observed in proteins and in water instead of one lifetime found in ethanol can be assigned to the high contact between TNS molecules. Our results are discussed in terms of solvent polarity and interaction within fluorophore molecules bound to proteins.     

硫杂杯芳烃基胶束自组装荧光化学传感器制备及其性能研究

以对叔丁基硫杂杯[4]芳烃（TCA）为受体、苝和十二烷基苯磺酸钠（SDBS）分别为荧光体和自组装模板剂,通过在水中的胶束自组装作用制备得到一种新型的“ON–OFF”型Cu2+荧光化学传感器。论文选用荧光猝灭率为考察指标,详细考察了受体TCA用量、自组装模板剂SDBS浓度、Cu2+浓度和共存金属离子等影响因素对胶束自组装荧光化学传感器Cu2+检测性能的影响情况。实验结果表明,当受体TCA与荧光体苝的摩尔浓度比值达到1 000,自组装模板剂SDBS浓度为50 mmol·L-1,该荧光化学传感器对水中铜离子具有较好的检测能力,待测Cu2+浓度在一定浓度范围内与荧光猝灭率呈线性相关。此外,荧光化学传感器的Cu2+检测性能基本不受Pb2+, Cd2+, Mn2+, Na+, K+, Ca2+, Mg2+, Al3+,Ni2+,Zn2+等共存金属离子干扰。该胶束自组装荧光化学传感器对Cu2+的选择性检测性能主要归因于胶束表面活性剂分子层中TCA受体对Cu2+的识别作用,而传感器荧光猝灭主要基于胶束内部的电荷转移或能量转移机制。     

Excitation-wavelength Dependent Fluorescence of Ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate

The excitation wavelength dependence of the steady-state and time-resolved emission spectra of ethyl 5-(4-aminophenyl)-3-amino-2,4-dicyanobenzoate (EAADCy) in tetrahydrofuran (THF) at room temperature has been examined. It is found that the ratio of the fluorescence intensity of the long-wavelength and short-wavelength fluorescence bands strongly depends on the excitation wavelength, whereas the wavelengths of the fluorescence excitation and fluorescence bands maxima are independent on the observation/excitation wavelengths. The dynamic Stokes shift of fluorophore in locally excited (LE) and intramolecular charge transfer (ICT) states has been studied with a time resolution about 30 ps. The difference between Stokes shift in the LE and ICT states was attributed to the solvent response to the large photoinduced dipole moment of EAADCy in the fluorescent charge transfer state. On this base we can state that, the relaxation of the polar solvent molecules around the fluorophore was observed.     

Studies of New Peroxyoxalate-H<Subscript>2</Subscript>O<Subscript>2</Subscript> Chemiluminescence System Using Quinoxaline Derivatives as Green Fluorophores

Quinoxaline derivatives are a great interest as fluorescent emitters for peroxyoxalate chemiluminescence. Reaction of peroxyoxalates such as bis-(2,4,6-trichlorophenyl) oxalate with H₂O₂ can transfer energy to fluorophore via formation of dioxetanedione intermediate. Two quinoxaline derivatives used as a fluorophore in this study which produce a green light in the chemiluminescence systems. The relationship between the chemiluminescence intensity and concentrations of fluorophore, peroxyoxalate, sodium salicylate and hydrogen peroxide was investigated. Kinetic parameters for the peroxyoxalate-chemiluminescence were also calculated from the computer fitting of the corresponding chemiluminescence intensity/time profiles. It was found that the biphenylquinoxaline can be used as an efficient green fluorescent emitter.     

Effect of a protein on the light energy conversion in dual fluorophore-nitroxide probes studied by ESR and fluorescence spectroscopy

Probing biological environment with dual fluorophore-nitroxide (FN) molecules in which fluorophore is tethered with nitroxide, a fluorescence quencher, opens unique opportunities to study molecular dynamics and micropolarity of the medium which affect intramolecular fluorescence quenching (IFQ), electron transfer, photoreduction and light energy conversion. In such molecules, the excited fragment of the chromophore can serve as an electron donor, and the nitroxide fragment as an electron acceptor. The same groups allow monitoring of molecular dynamics and also make it possible to measure micropolarity of the medium in the vicinity of the donor (by fluorescence technique) and acceptor (by electron spin resonance [ESR]) moieties. In the present work, two dual dansyl-nitroxide probes were incorporated in a binding site of bovine serum albumin. Their interactions with the protein, mobility, and photoreduction, as well as micropolarity of the media, have been studied by ESR and fluorescence methods. IFQ and spectral relaxation shift of the dansyl fragment have been monitored by time-resolved fluorescence technique. In parallel, computational studies on intramolecular dynamics of the FN probe were performed. On the basis of the Marcus model of the electron transfer between the excited dansyl fluorophore (donor) and nitroxide group (acceptor) and our experimental data, the mechanism of the electron transfer in the dual molecules incorporated into bovine serum albumin was established. It was shown that dual FN molecules in the protein meet main requirements for an efficient light energy conversion system.     

Time-resolved imaging of latent fingerprints with nanosecond resolution

Imaging of latent fingerprints using time-resolved (TR) method offers a broader platform to eliminate the unwanted background emission. In this paper, a novel TR imaging technique is demonstrated and implemented, which facilitates the detection of latent fingerprints with nanosecond resolution. Simulated experiments were carried out with two overlapping fingerprints treated with two fluorescent powders having different lifetimes in nanosecond range. The dependence of the fluorescence emission intensity in nanosecond resolution of TR imaging is also revealed.     

Time-resolved spectroscopic imaging reveals the fundamentals of cellular NADH fluorescence

A time-resolved spectroscopic imaging system is built to study the fluorescence characteristics of nicotinamide adenine dinucleotide (NADH), an important metabolic coenzyme and endogenous fluorophore in cells. The system provides a unique approach to measure fluorescence signals in different cellular organelles and cytoplasm. The ratios of free over protein-bound NADH signals in cytosol and nucleus are slightly higher than those in mitochondria. The mitochondrial fluorescence contributes about 70% of overall cellular fluorescence and is not a completely dominant signal. Furthermore, NADH signals in mitochondria, cytosol, and the nucleus respond to the changes of cellular activity differently, suggesting that cytosolic and nuclear fluorescence may complicate the well-known relationship between mitochondrial fluorescence and cellular metabolism.     

The Influence of Solvent Viscosity on the Fluorescence Decay and Time-Resolved Anisotropy of Green Fluorescent Protein

This report describes fluorescence decay and time-resolved anisotropy studies of green fluorescent protein (GFP) in various environments. The addition of glucose and fructose, NaCl, or polyethylene glycol changes the viscosity of the medium surrounding the GFP. Both the time-resolved anisotropy and the fluorescence decay of GFP are measured and it is shown that only the time-resolved anisotropy of GFP is affected by the viscosity, but not its fluorescence decay.     

Imaging Transparent Objects in a Turbid Medium Using a Femtosecond Optical Kerr Gate

A femtosecond optical Kerr gate time-gated ballistic imaging method is demonstrated to image a transparent object in a turbid medium. The shape features of the object are obtained by time-resolved selection of the ballistic photons with different optical path lengths, the thickness distribution of the object is mapped, and the maximum is less than 3.6%. This time-resolved ballistic imaging has potential applications in studying properties of the liquid core in the near field of the fuel spray.