Confocal fluorescence polarization microscopy for linear unmixing of spectrally similar labels

Studies of biological samples often call for simultaneous identification of multiple molecular or structural components. Multiple labelling fluorescence techniques are a powerful way of achieving this. However, the ability to distinguish a number of fluorescent probes unambiguously can be restricted by the fact that fluorescence spectra are generally broad and overlapping. Recently a technique known as linear unmixing has been combined with spectral imaging to discriminate between multiple fluorophores. In this study a scheme is proposed whereby fluorescence polarization information is used to expand the capability of the linear unmixing technique to accommodate additional fluorescent probes. As a proof-of-concept, it is shown that this polarization-based technique can be used to divide the signals generated by two spectrally similar fluorescent probes into their separate components.     

Near-infrared laser scanning confocal microscopy and its application in bioimaging

Near-infrared (NIR) fluorescence imaging is an important imaging technology in deep-tissue biomedical imaging and related researches, due to the low absorption and scattering of NIR excitation and/or emission in biological tissues. Laser scanning confocal microscopy (LSCM) plays a significant role in the family of fluorescence microscopy. Due to the introduction of pinhole, it can provide images with optical sectioning, high signal-to-noise ratio and better spatial resolution. In this study, in order to combine the advantages of these two techniques, we set up a fluorescence microscopic imaging system, which can be named as NIR-LSCM. The system was based on a commercially available confocal microscope, utilizing a NIR laser for excitation and a NIR sensitive detector for signal collection. In addition, NIR fluorescent nanoparticles (NPs) were prepared, and utilized for fluorescence imaging of the ear and brain of living mice based on the NIR-LSCM system. The structure of blood vessels at certain depth could be visualized clearly, because of the high-resolution and large-depth imaging capability of NIR-LSCM.     

结构光照明超分辨光学显微成像技术与展望

结构光照明显微镜(Structured Illumination Microscopy,SIM)通过结构化照明在频率域以空间混频的方式将物体高频信息载入光学系统的探测通带内实现突破衍射极限的超分辨光学显微成像。SIM凭借其较低的激发光强、对荧光染料的非特异性需求以及快速的宽场成像优势已成为活细胞超分辨光学显微成像方面应用最多的技术。本文系统回顾了SIM的技术进展,对SIM的基本原理与实现方法进了详细的分析,重点介绍了本课题组研发的基于光谱分辨的单光子激发超分辨显微镜和结合自适应光学的双光子激发超分辨显微镜这两种最新的SIM技术,最后简要讨论了SIM技术在生物成像中的应用及未来发展方向。     

Multispectral imaging with a confocal microendoscope

The concept of a multispectral confocal microscope for in vivo imaging is introduced. To demonstrate the concept we modified a slit-scan fluorescence confocal microendoscope incorporating a fiber-optic catheter for in vivo imaging to record multispectral images. The system was designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. Preliminary experiments were carried out in phantoms and cell cultures to demonstrate the potential of the technique.     

用于探测生物芯片的制冷型ICCD系统

利用光锥耦合的ICCD系统探测荧光染料标记的生物芯片,并对CCD芯片和像增强器制冷,以提高探测灵敏度。基于实验分析结果,指出背景噪声的主要来源为杂散光和生物芯片基底所发的荧光,指出用镜头成像限制了系统探测灵敏度的提高,可采用低荧光物质作为生物芯片的基底对系统加以改进。     

Single-molecule detection using continuous wave excitation of two-photon fluorescence

Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.     

Wavelength-Resolved 3-Dimensional Fluorescence Lifetime Imaging

We have developed a compact system for wide-field fluorescence imaging, resolved in three spatial dimensions, lifetime and wavelength, that is based on a gated optical intensifier and an all-solid-state diode pumped Cr:LiSAF oscillator-amplifier system. Exploiting spectral separation, the system has been applied to human teeth, obtaining good lifetime contrast between enamel, dentin and caries. Exploiting spectral separation combined with depth resolution, the study of fluorescent microspheres led to an enhancement in both lifetime contrast and lateral resolution.     

Spectral unmixing combined with Raman imaging,a preferable analytic technique for molecule visualization

This article reviews spectral unmixing used as a new method for Raman imaging. Raman spectroscopy explores the vibrational information about specific chemical bonds in molecules, which can be used for label-free molecular visualization. However, chemical bonds are usually shared among different molecules, which results in closed or mixed Raman peaks of many molecules. Therefore, the acquired spectra cannot be directly used to reconstruct the Raman images, as pure component spectra are hidden under the acquired spectra. Spectral unmixing is an effective method to provide a meaningful spectrum of each component with no priori spectral information and to also reconstruct the compositional distribution images. This article summarizes some representative spectral unmixing approaches used for Raman imaging and many related researches. This review strives to introduce the combination of spectral unmixing and Raman imaging as an efficient analytic technique to characterize various constituents and make subtle understanding of those complex structures.     

Metabolism-enhanced tumor localization by fluorescence imaging: in vivo animal studies

We present a high-sensitivity near-infrared optical imaging system for noninvasive cancer detection and localization based on molecularly labeled fluorescent contrast agents. This frequency-domain system utilizes the interferencelike pattern of diffuse photon density waves to achieve high detection sensitivity and localization accuracy for the fluorescent heterogeneity embedded inside the scattering media. A two-dimensional localization map is obtained through reflectance probe geometry and goniometric reconstruction. In vivo measurements with a tumor-bearing mouse model by use of the novel Cypate-mono-2-deoxy-glucose fluorescent contrast agent, which targets the enhanced tumor glycolysis, demonstrate the feasibility of detection of a 2-cm-deep subsurface tumor in the tissuelike medium, with a localization accuracy within 2-3 mm.     

用光子标记技术实时动态观测中性粒细胞吞噬病原体细胞

基因标记和光子学显象技术相结合,实时观测中性粒细胞吞噬病原体细胞骨架运动规律。利用致冷CCD系统组建了高灵敏度的荧光激发与测试装置。将带有绿色荧光蛋白(GFP)基因的PRSETB质粒转化大肠杆菌BL21,利用BL21高效表达GFP后发出强烈绿色荧光的特点,用光子显象技术连续记录荧光的变化,从而在不受干扰的条件下实时动态观察了中性粒细胞粘附、内吞、消化大肠杆菌的全过程。     

一种月壤主要矿物组分含量反演的光谱解混方法

从月壤光谱中挖掘矿物含量信息是月球遥感探测的重要内容之一,矿物光谱混合规律复杂,光谱特征对测试环境、粒度、地形、背景极为敏感是难点。提出并试验了一种基于光谱分解反演矿物含量的方法。在光谱分解前采用光谱连续统去除突出光谱谱形,分解中综合运用了光谱反射峰(介于两个吸收谷之间的光谱曲线向上凸起的部分)与吸收谷,分解后采用Hapke模型修正光谱非线性混合的影响,在混合端元光谱、端元化学成分与粒度未知情况下,实现了对月壤四种主要矿物橄榄石、斜方辉石、单斜辉石、斜长石的43条混合光谱的盲分解与矿物含量反演,反演的残差、误差、线性拟合相关系数(反演含量与真实含量)均值分别为5.0 Vol%,14.4 Vol%,0.92。该方法与反演结果可为月球高光谱矿物识别与填图提供参考与依据。     

超分辨率成像荧光探针材料应用进展

为了进一步认知复杂环境中的细胞生物学过程,研究人员发展了各种各样的生物成像技术。在这些技术中,生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而,传统的荧光成像技术受到了光学衍射极限的限制,无法分辨低于200 nm的空间结构,阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制,能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外,目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。     

Method for In-Vivo Fluorescence Imaging Contrast Enhancement through Light Modulation

Early diagnosis is one of the most important factors that increase the therapeutic potential of the disease. Diagnoses conducted by conventional equipment are expensive, time-consuming, burdensome to patients, and do not have high success rates. Diagnostic methods have also been investigated using nanoparticles. However, there have been no significant improvements in the early diagnosis of disease. The diagnosis technique proposed in this paper consumes less time, is more cost-effective, and more accurate. It uses a new concept—a low-intensity fluorescence molecular imaging system with a lock-in technique. This study applied the lock-in technique to basic research in contrast enhancement and optimization. This improved fluorescence distribution analysis, resulting in increased resolution of optical molecular imaging for early diagnosis of disease. An experimental lock-in fluorescence imaging system, which used a variety of fluorescent dyes, achieved signal amplification 100 times greater than that of a conventional fluorescence imaging system. The results of this study demonstrate that the lock-in technique could significantly improve optical molecular imaging technology, making it possible to achieve early diagnosis of disease.     

基于SiPM单光子探测器的荧光光谱仪研究

为了解决激光诱导荧光检测系统存在的光学结构复杂、体积大、成本高以及灵敏度不足等主要问题,研制了一种高灵敏、小型化的荧光光谱仪。该光谱仪以349 nm半导体激光器作为激发光源,采用正交型光路,将4×4窄带滤光片阵列与具有单光子灵敏度的硅光电倍增器（SiPM）阵列耦合,可实现光谱信息的多通道探测,具备结构紧凑、成本较低以及稳定性好等优点。以荧光素钠为测试样品,对光谱仪性能进行了评估。实验结果表明,光谱仪的检测限优于5×10?11 mol·L?1,在5×10?11 mol·L?1到1×10?9 mol·L?1的溶液浓度范围内,被测溶液浓度与检测所得荧光强度满足良好的线性关系,线性相关系数为0.998 39。此外,光谱仪还具备良好的重复性,荧光峰值强度的相对标准偏差小于10%。因此,该光谱仪兼具灵敏度高、线性度好、重复性与可靠性强等优点,可以满足现场实时检测的需求。     

基于超表面的多光谱成像系统设计

超表面是一种人工制造的亚波长结构阵列平面,重量轻,易集成,可实现多种功能,被广泛应用于诸多领域。传统光谱成像系统依赖于色散元件及光程累积相位差实现不同波长的色散与聚焦,无法满足系统集成化需求。不同于传统光学元件依赖电磁波在介质中传播累积相位差,超表面依靠界面相位变化来进行相位调控,可实现十分轻薄的光学系统。研究传输相位型超表面,使用时域有限差分算法(FDTD算法)优化单元结构。将超表面引入光谱成像系统中,通过优化亚波长结构尺寸,进行结构排布,开展超表面光谱成像系统研究,实现多波长色散与聚焦独立调控。利用该方法,扫描不同单元结构参数对相位的影响,依照聚焦的相位分布针对不同波长设计对应的位相分布,仿真实现了一个波段范围为510～720 nm,焦距为2 mm,谱段数为八个的超表面多光谱成像系统。通过电磁仿真软件FDTD solutions和数据处理软件计算全模结构电场的远场分布,并分析了系统的成像性能。相比于传统光栅或棱镜分光结构,超表面光谱成像系统可有效减小系统体积,其超轻、超薄、便携特点解决了现有光谱成像系统的应用局限性,为小型化、轻量化光谱成像系统的研制提供了一种新的解决方案。     

CCD紫外增强薄膜旋涂法工艺优化

在硅基探测器的入射窗上制备荧光下转换薄膜,是一种有效降低成本的紫外荧光增强技术。从理论上探讨了由聚二甲基硅氧烷与颜料黄101混合胶体的紫外荧光薄膜旋涂工艺参数与性能之间关系,搭建紫外荧光薄膜应用于光谱分析的性能测试实验平台,对紫外荧光增强薄膜旋涂工艺参数质量配比、旋涂转速进行优化。光谱分析探测器有两个主要指标,光谱响应灵敏度和光谱分辨率,分析与实验结果表明,利用旋涂法制备紫外增强荧光薄膜,旋涂转速将直接影响薄膜的厚度、表面粗糙度和荧光物质的分布,从而影响光谱分析系统的分辨率;紫外荧光增强薄膜的增强效率与荧光溶剂聚二甲基硅氧烷与荧光物质颜料黄101的质量比密切相关,质量比低无法满足对紫外响应效率的提高,但高质量比,荧光物质处在聚集态荧光自猝灭严重,也不利于增强薄膜的紫外响应效率。最终,在薄膜旋涂工艺优化的基础上,旋涂转速2 500～3 000 r·min-1,荧光物质与荧光溶剂质量比为7∶100制备出紫外荧光增强薄膜。汞灯特征光谱测试结果表明该薄膜313 nm紫外波长处探测响应灵敏度提高了1.6倍左右,对比分析镀膜前后特征光谱的半波带宽,镀制紫外增强荧光薄膜对其影响很小。     

双光子荧光与相干反斯托克斯拉曼散射显微成像技术的实验研究

双光子荧光与相干反斯托克斯拉曼散射同属于三阶非线性效应,二者之间的差异与联系是一个值得研究的问题.本文基于自行搭建的超连续谱近红外宽带相干反斯托克斯拉曼散射显微成像系统进行光谱成像,同时通过理论与实验对比分析了双光子荧光与相干反斯托克斯拉曼散射图像存在差异的原因.结果表明,具有亚微米以上横向分辨率的相干反斯托克斯拉曼散射成像系统,可以使用较大尺寸的荧光珠进行双光子荧光成像,通过解卷积得到双光子荧光成像的系统分辨率,并将它近似等效于相干反斯托克斯拉曼散射成像系统的当下分辨率.如果需要得到相干反斯托克斯拉曼散射成像系准确的分辨率结果,就必须使用尺寸比相干反斯托克斯拉曼散射成像系统实际分辨率小的球形样品进行实验测量.     

基于荧光光谱法的钞票识别技术

以光栅光谱仪为基础,光电倍增管作为探测器,运用VC++语言设计了计算机采样界面,形成了荧光光谱分析的钞票识别光电系统。在紫光灯激发下,对钞票进行了荧光光谱特性分析,设计了基于荧光光谱分析的钞票识别光学系统、光电信号的检测和信号处理系统。测试结果表明：真假币的荧光光谱曲线有着明显的区别,它们的峰值分别为545nm和525nm。设计的荧光光电系统灵敏度高,可靠性好。     

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,...