Fast fluorescence lifetime imaging techniques:A review on challenge and development

Fluorescence lifetime imaging microscopy(FLIM)is increasingly used in biomedicine,material science,chemistry,and other related research fields,because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments,studying interaction between proteins,metabolic state,screening drugs and analyzing their efficacy,characterizing novel materials,and diagnosing early cancers.Understandably,there is a large interest in obtaining FLIM data within an acquisition time as short as possible.Consequently,there is currently a technology that advances towards faster and faster FLIM recording.However,the maximum speed of a recording technique is only part of the problerm.The acquisition time of a FLIM image is a complex function of many factors.These include the photon rate that can be obtained from the sample,the amount of information a technique extracts from the decay functions,the fficiency at which it determines fluorescence decay parameters from the recorded photons,the demands for the accuracy of these parameters,the number of pixels,and the lateral and axial resolutions that are obtained in biological materials.Starting from a discussion of the parameters which determine the acquisition time,this review will describe existing and emerging FLIM techniques and data analysis algo-rithms,and analyze their performance and recording speed in biological and biomedical applications.

一种对高速运动目标的单光子测距方法

作为一种前沿的激光探测技术,单光子激光测距技术已成功应用于月球测距、卫星测距和地面测高等领域。然而,单光子测距在机载空对空、地对空平台上对高速运动目标进行跟踪测距时,回波光子落在不同的时间窗,导致直接计数无法有效提取信号的问题仍需解决。针对空对空条件下单光子激光测距的应用需求,基于时间相关光子计数技术设计一种适用于全天时、宽时域、多噪声条件下对高速运动目标的单光子测距方法。该方法采用阵列单光子探测器和相邻时间窗相关统计多帧处理算法提取激光回波光子信号,并在Matlab平台上对算法进行仿真实验,使用多元阵列单光子探测器实现最大测程百公里以上、背景噪声计数率约为5 MHz、单脉冲回波光子计数平均值为1条件下的回波光子信号提取。该方法能够克服传统单光子探测只能对准静态目标测距,只能在小接收视场和小波门范围等弱背景噪声及目标轨迹可预测条件下应用的限制,将单光子探测由只能固定平台夜晚对准静态目标测距推广至通用平台全天时对高速运动目标测距。

Single-photon microwave photonics

With the rapid development of microwave photonics technology, high-speed processing and ultra-weak signal detection capability have become the main bottlenecks in many applications. Thanks to the ultraweak signal detection capability and the extremely low timing jitter properties of single-photon detectors, the combination of single-photon detection and classical microwave photonics technology may provide a solution to break the above bottlenecks. In this paper, we first report a novel concept of singlephoton microwave photonics(SP-MWP), a SP-MWP signal processing system with phase shifting and frequency filtering functionalities is demonstrated based on a superconducting nanowire single photon detector(SNSPD) and a successive time-correlated single photon counting(TCSPC) module.Experimental results show that an ultrahigh optical sensitivity down to-100 d Bm has been achieved,and the signal processing bandwidth is only limited by the timing jitter of single-photon detectors. In the meantime, the proposed system demonstrates an ultrahigh anti-interference capability, only the signal which is phase locked by the trigger signal in TCSPC can be extracted from the detected signals combining with noise and strong interference. The proposed SP-MWP concept paves a way to a novel interdisciplinary field of microwave photonics and quantum mechanism, named by quantum microwave photonics.

基于模板的光辐射分析攻击

根据密码芯片光辐射的数据相关性,利用时间相关单光子计数技术搭建了光辐射分析实验平台,对基于模板的光辐射分析攻击进行分析。通过对光辐射迹进行模板刻画、构建策略和匹配方法的讨论,结合密码芯片光辐射的数据相关性,采用基于光辐射汉明重量模型的方法进行模板构建,并选取高级加密标准(AES)加密算法第一轮加密的S盒输出作为攻击点,结果表明基于该方法的模板匹配相较汉明距离等其他模型具有更加精确的匹配度。成功实施基于模板的光辐射分析攻击。

基于时间相关单光子计数的非接触式荧光层析成像系统

基于时间相关单光子计数(TCSPC)技术原理,研究了一套面向早期乳腺癌检测的时域荧光层析成像系统.系统采用非接触式空间光扫描测量方式,相对于传统的光纤接触式测量方法,可增大空间采样量、减小测量误差、提高空间分辨率.通过仿体实验对系统的成像质量进行了验证,结果表明:单目标仿体实验能较好地对荧光产率与寿命进行重建;在双目标仿体荧光产率的重建中,可有效对中心距为20 mm、边距为15 mm的目标体进行分辨,且对不同浓度荧光剂目标的重建具有较好的线性.实验结果证明系统应用于乳腺检测的可行性,进一步发展可有望应用于临床乳腺成像中.

基于透射解析模型的时域扩散荧光层析原理与实验

利用基于耦合扩散方程拉氏变换的广义脉冲谱技术和基于玻恩比的逆模型归一化表示形式,提出了无限平板透射模式下的时域扩散荧光层析图像重建方法.该方法采用外推边界条件下无限平板结构扩散方程的解析解,通过引入一对实拉氏变换因子及相应的两次线性求逆,可实现荧光产率和寿命的同时重建.将提出的算法进行了数值模拟验证,在空间分辨率、噪声鲁棒性等方面进行了评估.通过实验验证获得的荧光参数空间分布较准确的重建结果证明了此重建算法的有效性和可行性.

铝离子对辅酶NADH分子荧光增强和构象变化的影响

烟酰胺腺嘌呤二核苷酸(NADH)是生物体内重要的辅酶分子,在细胞能量代谢中发挥着关键作用。金属离子可以影响NADH所参与的酶促反应,其中铝离子(Al^(3+))对神经系统具有毒性,可以引发神经退行性疾病。因此,Al^(3+)和NADH分子间相互作用的研究有助于了解Al^(3+)对生物体内TCA循环和酶促反应的影响,具有重要的生物学意义。本文采用紫外-可见吸收和稳态荧光光谱,结合时间相关单光子计数技术(TCSPC),研究了Al^(3+)对水溶液中NADH的本征荧光光谱和分子构象变化的影响。紫外-可见吸收光谱显示,NADH与Al^(3+)的结合不会改变NADH分子腺嘌呤和烟酰胺两个本征发色团的吸收特性。为避免NADH分子内两个本征发色团之间的荧光共振能量转移效应的影响,采用340 nm作为激发波长,比较了NADH与Al^(3+)作用前后的荧光特性。实验结果证实,Al^(3+)可以与NADH焦磷酸盐桥上的两个氧原子相结合,使NADH分子的结构变得相对更加刚性,从而抑制NADH分子在溶液中的转动等非辐射过程,导致NADH分子平均荧光寿命增加,最终引起NADH分子荧光强度随Al^(3+)浓度的增加而线性增强。进一步,采用NADH本征荧光寿命振幅比的研究方法表征了NADH分子在溶液中的两种主要构象形式:腺嘌呤和烟酰胺相互堆积的折叠构象以及腺嘌呤和烟酰胺相互分离的展开构象。研究发现,Al^(3+)会打破溶液中NADH分子展开构象和折叠构象的平衡状态,促使辅酶NADH分子的展开构象转变为折叠构象,最终达到新的动态平衡,并且当NADH和Al^(3+)以不大于1∶2的浓度比结合时,NADH分子两种构象的振幅比与铝离子浓度的对数间存在线性关系,在Al^(3+)浓度检测等领域具有良好的应用前景。

Ultra-low power anti-crosstalk collision avoidance light detection and ranging using chaotic pulse position modulation approach

A novel concept of collision avoidance single-photon light detection and ranging（LIDAR） for vehicles has been demonstrated, in which chaotic pulse position modulation is applied on the transmitted laser pulses for robust anti-crosstalk purposes. Besides, single-photon detectors（SPD） and time correlated single photon counting techniques are adapted, to sense the ultra-low power used for the consideration of compact structure and eye safety. Parameters including pulse rate, discrimination threshold, and number of accumulated pulses have been thoroughly analyzed based on the detection requirements, resulting in specified receiver operating characteristics curves. Both simulation and indoor experiments were performed to verify the excellent anti-crosstalk capability of the presented collision avoidance LIDAR despite ultra-low transmitting power.

Terpyridine-derived Zn^(2+) Selective Fluorescence Sensor: Ratiometric Fluoroionophoric Behavior Study

A sensitive and selective zinc ion ratiometric fluorescence sensor has been synthesized and characterized. This material displays dual fluorescence. After the material was bonded to a closed-shell metal ion, such as Zn2＋, the recovery of the local excited fluorescence of the material-Zn2＋ complex, largely at the expense of the intramolecular charge transfer fluorescence, is consistent with the difference between selected orbital transitions of the free dye and the metal-chelated complex. For instance, the contribution of the πtpy→πtpy, transition becomes more prominent. This is also consistent with the results of the fluorescence decay behavior, measured via a time-correlated single photon counting setup. In contrast, the corresponding open shell Ni2＋ -bound complex quenches both kinds of photoluminescence, due to spin-orbit coupling.

Resolution and contrast enhancement of laser-scanning multiphoton microscopy using thulium-doped upconversion nanoparticles

High-contrast optical imagi ng is achievable using phosphoresce nt labels to suppress the short-lived background due to the optical backscatterand autofluoresce nee.However,the long-lived phosphorescence is generally incompatible with high-speed laser-scan ning imaging modalities.Here,we show that upc on versi on nan oparticles of structure NaYF4:Yb co-doped with 8%Tm(8T-UCNP)in combi nation with a commerciallaser-scanning multiphoton microscopy are uniquely suited for labeling biological systems to acquire high-resolution images with the enhancedcon trast.In comparison with many phosphoresce nt labels,the 8T-UCNP emission lifetime of-15μs affords rapid image acquisition.Thehigh-order optical nonlinearity of the 8T-UCNP(n=4,as confirmed experimentally and theoretically)afforded pushing the resolution limitattain able with UCNPs to the diffraction-limit.The contrast enha nceme nt was achieved by suppressing the backgro und using(i)ban dpassspectral filtering of the narrow emission peak of 8T-UCNP at 455-nm,and(ii)time-gating implemented with a time-correlated single-photon counting system that demonstrated the contrast enhancement of>2.5-fold of polyethyle neimine-coated 8T-UCNPs take n up by huma nbreast adeno carcinoma cells SK-BR-3.As a result,discrete 8T-UCNP nan oparticles became clearly observable in the freshly excised splee ntissue of laboratory mice 15-min post in trave nous injectio n of an 8T-UCNP solution.The dem on strated approach paves the way forhigh-contrast,high-resoluti on,and high-speed multiphot on microscopy in challe nging envir onments of i ntense autofluorescence,exogenous staining,and turbidity,as typically occur in intravital imaging.

荧光寿命测定的现代方法与应用

介绍了时间相关单光子计数、相调制和频闪等三种现代荧光寿命测定方法的工作原理 ,指出了各种方法的优点和局限性 ;介绍了时间相关单光子计数实验数据的处理方法 ;概述了时间分辨荧光技术在化学和生命科学中的应用。