Estimating the depth and lifetime of a fluorescent inclusion in a turbid medium using a simple time-domain optical method

A simple time-domain optical method for estimating the depth (d) and lifetime (tau) of fluorescent inclusions in a turbid medium is described. We demonstrate the method for depth and lifetime estimation of a fluorescent inclusion directly by fitting a monoexponential decay (tau(eff)) of the temporal position of the temporal point-spread function and the measurement of its maximum temporal position (t(max)). Since both of these measurements are intensity independent, this method provides a robust and efficient approach. This method is validated with experimental data.     

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.     

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.     

丹酰氯SiO2纳米发光标记物的制备

通过合成丹酰氯的荧光单体硅酯前驱物,采用油包水的反相微乳液法,以丹酰氯的荧光单体硅酯前驱物为核材料,成功地制备了丹酰氯的荧光纳米颗粒,克服了传统方法制备核壳荧光纳米颗粒中存在的荧光染料泄漏问题.通过透射电子显微镜表征该纳米粒子呈球形,大小均匀,直径为40nm左右.所制得纳米颗粒荧光性质稳定,受外界环境的影响小,且潜在生物亲和性,是一种新型的荧光标记物.     

光子计数激光雷达的时间相关卡尔曼深度估计

在高背景噪声和低积分时间的激光雷达远距离成像场景中,针对传统方法得到的深度图像目标被噪声淹没和深度估计偏差较大的问题,提出了一种基于信号光子时间相关性和自适应卡尔曼滤波器的深度信息估计方法。首先,提取在时间上具有聚集特征的光子计数形成集合;然后,分析了影响信号光子在时间上分布的因素并使用静态高斯线性模型来描述该集合;最后将集合中的所有光子飞行时间乱序,输入改进的自适应卡尔曼滤波器,从而迭代估计深度值。在信号噪声比为1的室内,积分时间分别为10 ms和1 ms时,本文方法相对传统的最大似然方法在均方根误差指标上提升了40%和38%。在信噪比约为0.135的室外2 km目标成像实验中,在信号光子数分别为100、33和17的情况下,本文方法成像效果都优于传统最大似然估计方法和时间相关光子快速去噪方法,得到的深度图像都更清晰,噪声更低。在高噪声和短积分时间下,本文方法可以被运用于激光雷达远距离成像的深度信息估计和图像恢复中。     

Relationship between depth of a target in a turbid medium and fluorescence measured by a variable-aperture method

The relationship between the depth of a target in a turbid medium and the fluorescence ratio profile measured by use of illumination and collection apertures with variable diameters and the same optical path is shown. The forward problem was studied by Monte Carlo simulations of the propagation of fluorescent light through a theoretical model of a biologically relevant system for a range of aperture diameters. The curve of the fluorescence ratio as a function of the aperture diameter is characterized by a maximum/minimum point whose position shifts linearly with the depth of the target. Furthermore, the position of the maximum/minimum is observed to be insensitive to variations in the fluorescence efficiency and to the optical properties of the target layer or the entire medium.     

Fiber-optic sensors for the estimation of oxygen gradients within biofilms on metals

This paper presents an optical approach to measure the oxygen gradients in biofilms grown on stainless-steel 316L. The sensor is constructed by immobilizing an oxygen-quenchable fluorophore at the tip of an optical fiber. The response time and reversibility of fiber-optic sensor have been analyzed. By mounting the optical fiber using a computerized micromanipulator, the vertical profiles of oxygen are measured with 10 μm depth resolution across biofilms on metal coupons. Steep concentration gradients of oxygen have been observed within the biofilm, both perpendicular and parallel to the metal surface. Optical response of the biofilm grown metal surface is found to be a consistent measure of the oxygen profiles within biofilms.     

Digital pH Fluorescent Sensing Shown by Small Organic Molecules

A series of 2-(N-oxide pyridyl)-5-methoxythiazoles were synthesized and corresponding optical properties were also investigated. It is first found that a digital-type fluorescent responses in a sharp pH variation with dual-emission could be fulfilled based on a small fluorophore.     

25 nm resolution single molecular fluorescence imaging by scanning near-field optical/atomic force microscopy

High-resolution single molecular near-field fluorescence images were observed by scanning near-field optical/atomic force microscopy (SNOM/AFM). We modified the SNOM/AFM for both high-resolution fluorescence imaging and high-resolution topographic imaging. The imaged fluorophore, Alexa 532, is prepared with a poly-methyl-methacrylate (PMMA) film coating. A fluorescence resolution of 25 nm was obtained with a simultaneous topographic image of a flat surface. A sample prepared with a lower PMMA concentration exhibited a rough surface in the micro area. The results for the flat surface indicated that the fluorescence resolution is worst in the rough surface sample, that the maximum fluorescence intensities for the individual fluorophore are similar, and that the decay rate is faster. Thus, we concluded that the morphological effect is an important factor in fluorescence image resolution and the apparent lifetimes of the fluorescence molecules.     

Lateral modulation boosts image quality in single plane illumination fluorescence microscopy

A new microscope combines optical sectioning by fluorophore excitation using a single light sheet with structured illumination. Several images with laterally intensity-modulated light sheets are recorded from scattering fluorescent specimens. By applying a simple data processing scheme, the nonmodulated volumes are identified. The blurred features become dark, and the resultant images are improved in terms of contrast and resolution. Hence, the instrument is capable of discriminating against contributions to the image that are induced by the optical properties of the specimen. The new microscope's capabilities are demonstrated by imaging the internals of the head of an adult Drosophila melanogaster (fruit fly) expressing green fluorescent protein-labeled polycomb proteins.     

压缩光场重建与深度估计

针对光场深度估计过程中数据量大、边缘处深度估计结果不准确问题,利用压缩感知原理重建光场,提出一种新的多信息融合的光场图像深度估计算法。利用压缩感知重建算法重建5×5视角光场数据,获取光场数据后首先移动子孔径实现重聚焦,然后利用角度像素块散焦线索和匹配线索计算出场景初始深度和置信度。计算图像边缘信息,通过融合初始深度、置信度、边缘信息获取最终深度。实现压缩光场仿真重建,并对仿真光场数据和公开光场数据进行深度估计,实验结果表明:可以仿真重建出5×5视角光场数据,且仿真重建的光场可用于深度估计。该深度估计算法在场景边缘处的深度估计结果边界清晰,层次分明,验证了重建光场深度估计的可行性与准确性。     

拖曳阵被动合成孔径目标深度稳健估计

使用水下无人平台作为载体的拖曳阵进行被动目标深度估计具有灵活性高和隐蔽性好的优点,针对实际应用中存在的平台自噪声和阵列瞬时随机加速度扰动问题,提出了一种稳健的目标深度估计方法。该方法分为三个步骤,首先对阵元接收信号进行自适应噪声抵消和相位抖动滤波,然后对声压进行距离积分实现简正波模态估计,最后计算模态匹配度,最大值对应的深度为目标深度估计结果。仿真表明在干扰背景下该方法的目标深度估计稳健性优于传统方法,声源频率、合成孔径距离和信干比决定了目标深度估计误差。利用实验数据验证了该方法对水下低频线谱声源的深度估计能力。     

硫杂杯[4]芳烃基胶束自组装荧光探针的结构性质及检测性能的研究

水体中重金属污染因威胁生态环境和人类健康而被受广泛关注。荧光探针由于具有快速高效检测重金属的特性,一直是该领域的研究热点。通常,荧光探针在结构上包括对待测物质起识别作用的受体和能产生信号响应的荧光体,并逐步形成了内在型、共轭型、系综型和模板辅助自组装型等四种结构类型。近年来,基于受体和荧光体在表面活性剂胶束内自组装而形成的胶束自组装型荧光探针因结构简单、易于制备、能直接应用于水环境等特点逐渐受到重视。以对铜离子具有优异结合性能的对叔丁基硫杂杯[4]芳烃（TCA）为受体,以芘、荧蒽、蒽、菲、苝等分子为荧光体,通过表面活性剂胶束自组装制备针对Cu2+检测的胶束自组装型荧光探针,采用参比法测定了胶束自组装荧光探针的荧光量子产率,采用稳态荧光法测定了胶束聚集数,同时通过计算荧光猝灭率分别考察了荧光体种类、复配表面活性剂等因素对该探针的Cu2+检测性能的影响情况。实验结果显示,采用十二烷基硫酸钠（SDS）、曲拉通100（TX-100）、聚氧乙烯月桂醚（Brij35）等三种不同的表面活性剂对探针荧光体的荧光量子产率产生了明显影响,测得的荧光探针荧光量子产率介于0.25~0.47,且三者逐渐增大,说明表面活性剂改变了胶束内荧光分子芘所处微环境的极性,且不同类型表面活性剂对微环境极性的影响程度有所差异,微环境极性的增强对极性更大的激发态芘具有更强的稳定作用。而受体TCA的加入对荧光体所处微环境极性影响较小,未对荧光量子产率产生较大影响。但TCA的加入使探针的胶束聚集数明显减少,这归因于具有两亲性的受体TCA分子通过胶束自组装进入并分散在表面活性剂分子层中,形成共胶束结构,从而改变了表面活性剂分子的聚集状态。荧光体变更对荧光探针的Cu2+检测性能有显著影响,在同样条件下,以荧蒽、蒽、菲作为荧光体的探针检测Cu2+所得到的荧光猝灭率远高于芘、苝,这主要是因为不同荧光体在从激发态返回基态时辐射跃迁所释放能量不同,其能量与受体TCA识别Cu2+所需能量之间的匹配度越高,荧光猝灭率越大。不同类型的表面活性剂之间的复配能明显提升荧光探针检测性能,当非离子/阴离子、非离子/阳离子型复配表面活性剂之间的复配比例分别为7∶3和1∶1时荧光猝灭率达到最大值,且均高于单一表面活性剂时的荧光猝灭率。这说明不同类型表面活性剂复配的最佳比例存在较大差异,但均有效地增强了受体与荧光体的分散性及自组装性能,提高了对Cu2+的检测性能。研究结果将为新型胶束自组装荧光探针的设计和应用提供数据参考。     

An analysis of dissolved organic matter from freshwater Karelian Lakes using reversed-phase high-performance liquid chromatography with online absorbance and fluorescence analysis

The spectral and optical properties of the fractionated components of dissolved organic matter (DOM) of three freshwater lakes in Karelia were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) with online detection of fluorescence and absorption spectra. It is shown that the DOM fractions are qualitatively similar, but differ quantitatively in the ratio of components and consist of at least three types of fluorophores: (1) hydrophilic “humic-like” fluorophore(s) with the emission maximum in the region of 420 nm and an absorption band at 260–270 nm; (2) hydrophobic “humic-like” fluorophore(s) with the emission maximum at approximately 450 nm that has no characteristic absorption maxima in the region from 220 to 400 nm; and (3) a “protein-like” fluorophore with the emission maximum in the region of 340–350 nm, which is typical of proteins and peptides containing tryptophan.     

Spectroscopic measurement of blood volume and its oxygenation in a small volume of tissue using red laser lights and differential calculation between two point detections

An optical method for measuring blood volume and its oxygenation in a small volume of tissue (<1 cm³) based on the Beer–Lambert law is proposed. Three red laser lights are used because the difference of absorption by oxy- and deoxy-haemoglobin is large, but the absorption of the light by tissue itself causes a large offset value. The scattered light was detected at two receiving points, and the differential calculation between the values obtained from the receiving points were examined to eliminate the offset value. The result shows that the blood volume in skin is obtained from the regression analysis without the offset value. This method was evaluated by using a simple model which contained real blood. The relationship between the concentration of erythrocyte and the output of this method shows good linearity. Also, measuring depth as a function of the distance between incident and receiving points was estimated by using plastic plates having similar optical properties to skin. The maximum measuring depth was almost linear to the distance.     

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。     

小分子荧光探针的光谱

运用紫外-可见光谱、荧光光谱法研究了以二烯酮为荧光团的1,5-双(4-羟基)-1,4-戊二烯-3-酮探针的光学性质.一系列pH滴定实验表明探针的紫外吸收和荧光光谱随溶液的pH值的改变而变化.同时,探针紫外吸收光谱发生明显的红移,荧光光谱强度也随之变化,在碱性条件下,相对荧光强度浮动较大.解离常数pK<,α>为9.25,...     

An investigation into the role of the optical detection set-up in the recording of cardiac optical mapping signals: A Monte Carlo simulation study

Photon scattering is known to distort the fluorescence signals recorded from optically mapped cardiac tissue. However, the contribution of the parameters which define the optical detection set-up has not been assessed. In this study, Monte Carlo (MC) simulations of photon scattering within ventricular tissue are combined with a detailed model of a tandem-lens optical detection apparatus to characterise (i) the spatial origin upon emission of photons recorded in voltage-sensitive fluorescence measurements of cardiac electrical activity (using the fluorescent dye di-4-ANEPPS) and how this affects signal distortion, and (ii) the role the detector characteristics could play in modulating signal distortion during uniform illumination and photon emission from tissue depth. Results show that, for the particular excitation/emission wavelengths considered (488 nm and 669 nm, respectively), the dimensions of the scattering volume during uniform illumination extend around 3 times further in the surface recording plane than in depth. As a result, fluorescence recordings during electrical propagation are more distorted when transmembrane potential levels differ predominantly in the surface plane than in depth. In addition, MC simulation results show that the spatial accuracy of the fluorescence signal is significantly limited due to photon scattering, with only a small fraction of the recorded signal intensity originating from tissue beneath the pixel (approximately 11% for a 0.25×0.25 mm pixel). Increasing pixel size increases this fraction, however, it also results in an increase in the scattering volume dimensions, thus reducing the spatial resolution of the optical system, and increasing signal distortion. MC simulations also demonstrate that photon scattering in cardiac tissue limits the ability of optical detection system tuning in accurately locating fluorescent emission from depth. Specifically, our results prove that the focal plane depth that yields maximum signal intensity provides an underestimation of the emission depth. In conclusion, our study demonstrates the potential of MC simulations of photon scattering in guiding the design of optical mapping set-ups to optimise performance under diverse experimental conditions.     

一种基于模态域波束形成的水平阵被动目标深度估计

水面水下目标分辨与识别一直是被动声呐探测领域的难题.利用一种水平阵模态域波束形成算法获得己知方位目标声源的各阶模态强度,将其与不同深度的各阶参考模态强度进行匹配,最终实现了对声源的深度估计.仿真结果表明,该算法可以在信噪比为-10 dB的情况下,用300Hz带宽的信号样本,实现对声源深度的有效估计.系统分析了不同参数和不同波导条件对该方法目标深度估计性能的影响.其中,阵元数越多,模态样本数越多,计算频段越宽,方位估计精度越高,有效阵长越长,深度估计的性能越好.阵元间距和波导深度的变化不会影响该方法的深度估计性能,并且该方法的深度估计性能在声速剖面、海底参数等波导条件存在扰动时具有鲁棒性.     

A CTRW-based model of time-resolved fluorescence lifetime imaging in a turbid medium

We develop an analytic model of time-resolved fluorescent imaging of photons migrating through a semi-infinite turbid medium bounded by an infinite plane in the presence of a single stationary point fluorophore embedded in the medium. In contrast to earlier models of fluorescent imaging in which photon motion is assumed to be some form of continuous diffusion process, the present analysis is based on a continuous-time random walk (CTRW) on a simple cubic lattice, the objective being to estimate the position and lifetime of the fluorophore. This can provide information related to local variations in pH and temperature with potential medical significance. Aspects of the theory were tested using time-resolved measurements of the fluorescence from small inclusions inside tissue-like phantoms. The experimental results were found to be in good agreement with theoretical predictions provided that the fluorophore was not located too close to the planar boundary, a common problem in many diffusive systems.