Quantitative imaging of mixing dynamics in microfluidic droplets using two-photon fluorescence lifetime imaging

Droplet-based microfluidic systems enable miniaturization of chemical reactions in femtoliter to picoliter volume compartments. Quantifying mixing dynamics of the reagents in droplets is critical to determine the system performance. In this Letter, we developed a two-photon excitation fluorescence lifetime imaging technique to quantitatively image the mixing dynamics in micro?uidic droplets. A cross/autocorrelation method was used to reconstruct a high-quality fluorescence lifetime image of the droplet. The fluorescence decay was analyzed for accurate determination of the mixing ratio at each pixel of the image.     

荧光寿命显微成像技术及应用的最新研究进展

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响,荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点,近年来已被广泛应用于生物医学等领域.然而,尽管FLIM的发明和发展已历经数十年时间,其在实际应用中仍然面临着许多挑战.例如,其成像分辨率受衍射极限限制,而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来,相关硬件和软件的快速发展及其与其他光学技术的结合,极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点,在此基础上概述了该技术的最新研究进展,包括其成像性能的提升和在生物医学应用中的研究现状,详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力,以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用,最后对其未来发展趋势进行了展望.     

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca²⁺-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca²⁺ concentration image using anin vitro-determined calibration curve. The Ca²⁺ concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

双光子阵列点激发同时多维荧光信息的处理

五维同时荧光信息显微成像方法是一种新的荧光信息获取技术，它采用了双光子阵列点激发方式.这一方法可同时获取激发阵列点每点荧光的位置信息、荧光光谱信息和荧光寿命信息，弥补了现有荧光检测技术的不同功能信息不具有同时性的缺陷.给出了从这种技术的复合信息中提取复合光谱几何强度结构图像、不同光谱几何强度结构图像、不同光谱寿命图像的方法.提出了一种激发荧光强度修正系数矩阵方法，消除阵列点激发光强不均匀对激发荧光强弱产生的不利影响，取得明显效果.实验对实际样品做了数据采集和处理，给出图像结果，表明处理的效果良好.对存在的问题也作了讨论. 关键词： 荧光信息处理 双光子 荧光光谱 荧光寿命     

Fluorescence lifetime imaging of oxygen in living cells

The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence lifetime imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can be described by the Stem-Volmer equation. This shows that fluorescence quenching by oxygen is a dynamic quenching process. In addition, it was demonstrated that the fluorescence lifetime of RTDP is insensitive to pH, ion concentration, and cellular contents. This implies that a simple calibration procedure in buffers can be used to quantify oxygen concentrations within cells. First fluorescence imaging experiments on J774 macrophages show a nonuniform fluorescence intensity and a uniform fluorescence lifetime image. This indicates that the RTDP is heterogeneously partitioned throughout the cells, while the oxygen concentration is constant.     

Confocal fluorescence lifetime imaging of free calcium in single cells

Ca²⁺ concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for free calcium and exhibit marked changes in their photophysical properties upon binding. The differences in the fluorescent lifetime of the probes can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within the cells. We describe the use of a fast fluorescence lifetime imaging method in combination with a standard confocal laser scanning microscope for the determination of Ca²⁺ concentrations in single rat cardiac myocytes using the intensity probe Calcium Green.     

油溶性CdSe量子点荧光探针直接检测农药水胺硫磷

基于农药水胺硫磷对油溶性CdSe量子点荧光猝灭的现象,建立了一种简单、快速、直接检测农药水胺硫磷的新方法。在最佳实验条件下,油溶性CdSe量子点的荧光猝灭程度与水胺硫磷浓度在2.30×10-7～1.09×10-5 mol·L-1范围内呈较好的线性关系,相关系数为0.999 9,对水胺硫磷的检出限为1.1×10-7 mol·L-1。本法已成功用于大米和小麦面粉样品中水胺硫磷农药残留的检测,加入回收率在93.3%～105.0%之间,结果满意。结合紫外-可见吸收光谱及时间分辨荧光光谱,探讨了水胺硫磷对油溶性CdSe量子点荧光猝灭的机理。研究结果表明,水胺硫磷能有效改变油溶性CdSe量子点的表面状态,增大了表面缺陷和非辐射重组的发生,从而使油溶性CdSe量子点的荧光猝灭。     

一种改进的基于OTSU自适应阈值的机器人数字路标图像模糊增强算法

在机器人路径规划中，机器人数字路标识别是很重要的，图像的预处理会影响识别结果。图像增强技术是提高预处理结果的一种有效方法，模糊图像增强算法是目前广泛使用的一种增强算法。针对Pal模糊图像增强算法在隶属函数的定义和渡越点选择上的缺点，提出了一种改进的模糊增强算法。本算法首先使用OTSU算子自动选择最佳阈值，解决渡越点需要人工设置的缺点，并消除选择的随机性。然后修改模糊增强算法的核心隶属函数式，解决了图像像素的低灰度值被硬性设置为0的缺陷，从而改善了图像信息损失的问题。最后，将改进的算法用于处理Pioneer Ⅲ机器人的数字路标图像。实验结果表明，与现有的模糊增强算法相比，提出的算法可以取得好的效果，且提高了运算速度，具有一定得实用性和推广性。     

Time-gated biological imaging by use of colloidal quantum dots

The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.     

铕-吡哌酸时间分辨荧光法检测DNA含量

在pH值为7.2的Tris-HCl缓冲溶液中,铕与吡哌酸反应形成配合物。该体系中加入鲱鱼精DNA分子作用后荧光强度显著增强,并且在一定浓度范围内,DNA浓度与其荧光强度呈良好的线性关系,据此建立了一种简单的测定DNA的时间分辨荧光分析新方法。考查了体系的时间分辨荧光光谱,通过与普通荧光光谱的对比突显了采用时间分辨荧光法的优势,并对反应条件进行了优化。该方法对DNA的检测限为0.03 mg·L-1(3σ),对浓度为4.0 mg·L-1的DNA进行11次平行测定,其相对标准偏差为0.3％。DNA的浓度在0.1～6.0 mg·L-1范围内与荧光强度呈良好的线性关系,线性方程为：ΔI=89.58c(mg·L-1)+0.920 5,线性相关系数r=0.999 6。此方法已应用于合成样品中DNA的测定,结果和加标回收率令人满意。     

An integrated phase-resolved fluorescence imaging system with sub-nanosecond lifetime resolution

In this paper, a novel configuration of an integrated phase-resolved (PR) fingerprint fluorescence imaging system is proposed and implemented. In this integrated PR imaging system, a current modulated 402 nm dual diode laser is proposed to be the light source, to obtain both high laser power and easy modulability. To estimate the lifetime resolution of this PR imaging system, a novel method of using distance-selective suppression of fluorescence signals from two identical fluorescing samples is proposed. Detailed theoretical and experimental analyses are presented. The experimental results demonstrate that this integrated PR imaging system has a lifetime resolution of 0.1 ns. Fingerprint detection experiments are also carried out using this system with latent fingerprints deposited on substrates of aluminum foil and currency.     

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochemical properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radio frequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is separated in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calculate in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.     

Gradient-norm-based histogram equalization taking account of HSV color space distribution

In the present paper, we propose an image contrast enhancement method that can enhance the contrast of a color image naturally by taking account of a color space shape. The proposed method realizes the natural enhancement based on two kinds of intensity histograms: a gradient-norm-based histogram and an ideal histogram derived from the shape of a color space. The former histogram is used to suppress over-enhancement in the flat regions of an image and the latter histogram is used to prevent the whole image from being darken. Concretely, the aforementioned intensity histograms are appropriately mixed into a histogram with a weight based on the average intensity of the input image. The contrast enhancement of the input image is realized using the cumulative histogram of the mixed histogram as an intensity transform function. To verify the validity of the proposed method, in experiments, the proposed method is applied to a variety of images and experimental results are evaluated qualitatively and quantitatively.     

Tb—吡哌酸的荧光体及尿和血清中吡哌酸含量的测定

本文报道了Ｔｂ－吡哌酸生成络合物,在紫外光照射下发生分子内能量传递使Ｔｂ产生特征荧光,其荧光强度与吡哌酸的浓度成线性关系,由此建立了一种灵敏,快速的分析嘴哌酸的方法。     

Study on the New Fluorescence Enhancement System of Tb –N-(2 - Pyridinyl) Ketoacetamide-Et<Subscript>3</Subscript>N-Zn and its Application

A sensitive fluorescence enhancement system was developed for the determination of zinc (II). The fluorescence intensity of the Tb- N- (2 - Pyridinyl) ketoacetamide (PKA) system was greatly enhanced by the addition of triethylamine (Et₃N) and zinc nitrate in the methanol solution. The excitation and emission wavelengths were 329 nm and 546 nm, respectively. Under optimal conditions, the fluorescence intensities varied linearly with the concentration of Zn²⁺ in the range of 8.0×10⁻⁷−5.0×10⁻⁶ M with a detection limit of 9.9×10⁻⁸ M. The interferences of some substances were described. This method was applied to the determination of amounts of Zn²⁺ in soybean, rice, and wheat, respectively. The results showed that the proposed procedure is a high selective, simple, and rapid method to the determination of Zn²⁺ ion. The mechanism of fluorescence enhancement was also studied.     

应用小波边缘检测技术分析荧光寿命显微像

研究了小波图象边缘检测技术在荧光寿命显微像分析中的应用，利用小波分析的多尺度特性进行了荧光寿命显微像的边缘提取，结果表明，这种方法非常有效。     

融合改进直方图PDE和二维Tsallis熵多阈值SAR分割

针对合成孔径雷达(SAR)图像中存在大量的相干斑噪声,对SAR图像进行分割易出现分割不精、边缘模糊等问题,融合改进的直方图PDE和二维Tsallis熵多阈值,提出了一种SAR图像分割算法。根据PDE直方图均衡化方法,将图像去噪与图像增强加权融合,利用各自权值调整去噪项与图像增强项;同时将二维Tsallis熵单阈值分割方法扩展到多阈值分割, 建立基于多阈值的选取方法,并引入萤火虫算法来求解最优阈值对,实现了二维Tsallis熵多阈值对去噪增强SAR图像的有效分割。仿真结果表明:与其他3种分割算法相比,该文算法在处理噪声大、灰度差值小的图像时具有较高的分割精度,PRI至少提升2.53%、VOI降低8.48%、GCE降低11.14%。     

肺癌荧光图象的伪彩色增强处理

近年来,作者开发了肿瘤荧光成象法用于诊断和定位早期支气管癌.由于所获得的荧光图象使用了象增强器进行增益放大,因此还消除了肺癌荧光图象的彩色信息.本文给出了一种荧光图象伪彩色增强处理的方法,并证实了伪彩色增强技术用于早期肺癌荧光诊断的可行性.经增强处理后,内镜医生只要根据彩色视频图象中与黑白荧光图象强度相对应的颜色就可以鉴别肺癌存在与否.同增强处理前的黑白图象相比,医生从伪彩色图象中获得了病灶内部结构、边缘效果和立体感方面的更多信息.临床结果表明可疑组织与正常组织之间获得了足够高的颜色衬比,有助于诊断早期肺癌病灶.     

短波长X射线衍射检测晶体材料内部缺陷的边界阈值法

在普通X射线源上进行X射线衍射断层扫描成像检测晶体材料内部缺陷的研究极少,短波长X射线衍射仪(short wavelcngth X-ray diffractometer,SWXRD)相比同步辐射装置,体积小巧、实用和维护费用低.在SWXRI)上进行X射线衍射断层扫描时,缺陷边界的确定直接影响成像的质量和缺陷的辨别.采用衍射强度阈值法对测试数据进行处理,使得缺陷边界清晰可辨.运用Gauss函数拟合测试数据探索各种因素对阈值大小的影响,解决阈值设定的难题.通过研究置于铝粉中的不同直径像质计对阈值大小的影响,发现阈值为基体强度的91%较合适.然后测试铝板上的细缝进一步验证了阈值法对缺陷边界的改善和阈值选择的准确性.