利用荧光探针Fluo-4检测胸腺细胞内钙离子浓度变化

利用荧光显微数码成像系统测量荧光探针Fluo-4荧光强度的改变,建立动态检测细胞内钙离子浓度的方法.用荧光探针Fluo-4/AM标记原代培养小鼠胸腺细胞,以KCL刺激胸腺细胞去极化,并打开细胞膜上电压依赖性钙通道,细胞内荧光强度会发生改变.利用荧光显微数码成像系统动态监测Fluo-4荧光强度的改变可分析计算钙离子浓度.本方法灵敏度高,能实时监测细胞内钙离子浓度的变化.     

基于荧光分子断层成像的多模成像系统研究进展

基于荧光分子断层成像（FMT）的多模成像系统已广泛应用于动物实验研究,它将现有的其他成像模态与FMT系统相融合,实现同时对被测生物解剖学结构、生理学功能和分子或细胞水平生物学活动的在体成像.首先介绍了单模FMT系统的发展历史和现状,并介绍了国内外基于FMT的多模系统的研究进展,着重介绍了文献报道的典型的FMT/CT、FMT/MRI和FMT/放射性核素成像系统的系统组成、工作原理、性能特点及实验应用.对基于FMT的多模成像系统的发展进行了展望.     

近红外荧光散射断层成像的研究进展

近红外荧光光学断层成像(FODT)是以合适的荧光探针作为标记物或对比剂，用特定波长的红光激发荧光染料，使其发出波长长于激发光的近红外荧光，通过测量媒质边界处有限点的荧光强度，考虑光子在组织中传播的散射特性，来重建出组织内部的荧光光学特性的分布图像以及组织光学参数。这种成像方式具有无电离辐射、染料稳定、可长期监测和设备简单、成本低等优点，在肿瘤检测、基因表达、蛋白质分子检测和药物受体定位等方面有着很大的应用潜力。在给出近红外荧光散射断层成像典型系统的基础上，详述了近红外荧光在组织中的频域传播模型和重建算法；介绍了两家研究机构在此领域的研究进展；讨论了将该成像方法应用于临床的进一步的发展方向。     

动态荧光分子成像技术研究进展

动态荧光分子成像技术能够描述荧光分子探针在生物体内吸收、分布以及排出的完整过程,是一种可以对生物体的生理、病理过程进行连续监测的动态成像技术.这项技术具有无电离辐射、成像速度快、灵敏度及特异度高、费用低廉等优点,在基础医学及临床医学研究中具有广阔的应用前景.从系统、算法和应用3个方面对动态荧光分子成像技术的研究进展进行了综述.     

荧光定量PCR快速诊断21三体综合征

目的　建立荧光定量PCR技术检测 2 1三体综合征。方法　采用PCR方法同时扩增位于 2 1号染色体上的人肝型磷酸果糖激酶基因 (humanliver typephosphofructokinasegene ,PFKL CH 2 1)和位于 1号染色体上的人肌型磷酸果糖激酶基因 (humanmuscle typephosphofructokinasegene ,PFKM CH1) ,使用SYBRGreenⅠ荧光染料处理产物、琼脂糖电泳后在凝胶成像系统进行分析 ,得出扩增产物的荧光强度对比值。用此方法检测 2 6例 2 1三体综合征患儿及 2 0名正常人。结果　 2 6例 2 1三体综合征患儿PFKL CH2 1/PFKM CH 1扩增产物的荧光强度对比值为 1.5 8± 0 .17,而正常人为 1 0 0± 0 .0 5 ,两者差异有显著性。结论　SYBRGreenⅠ荧光定量PCR技术检测 2 1三体综合征具有准确、快速、安全、实用等特点 ,有较高的临床使用价值。     

荧光分子断层成像系统的研究进展与比较

荧光分子断层成像（FMT）利用具有特异性的荧光分子探针,在体成像、观测生物体内细胞和分子水平的变化。介绍FMT系统的国内外研究进展,从系统的复杂性、投影数目、光源-探测器（S-D）数据对集大小、重构图像的质量等方面,分析5种S-D几何结构的特点。着重从实现方面,分析、比较已报道的几种FMT系统的特点,并对其多模态成像的可扩展性、临床应用的可能性等进行了总结与展望。     

近红外荧光散射断层成像的研究进展

近红外荧光光学断层成像(FODT)是以合适的荧光探针作为标记物或对比剂,用特定波长的红光激发荧光染料,使其发出波长长于激发光的近红外荧光,通过测量媒质边界处有限点的荧光强度,考虑光子在组织中传播的散射特性,来重建出组织内部的荧光光学特性的分布图像以及组织光学参数。这种成像方式具有无电离辐射、染料稳定、可长期监测和设备简单、成本低等优点,在肿瘤检测、基因表达、蛋白质分子检测和药物受体定位等方面有着很大的应用潜力。在给出近红外荧光散射断层成像典型系统的基础上,详述了近红外荧光在组织中的频域传播模型和重建算法;介绍了两家研究机构在此领域的研究进展;讨论了将该成像方法应用于临床的进一步的发展方向。     

基于FRET荧光蛋白探针的活体定量分子影像方法探究

目的荧光共振能量转移(fluorescent resonance energy transfer,FRET)探针由于自身仍存在诸多局限,其在活体成像领域尚未得到广泛应用。本文旨在解决FRET探针在活体定量分子影像应用中所存在的瓶颈问题,即如何利用FRET探针精准计算目标物浓度,以及如何基于FRET探针实现三维成像。方法基于探针和目标物结合时的化学反应平衡关系,提出一种新型分析方法,以精确定量待测物[钙调素(calmodulin,Ca M)]的浓度。同时对于FRET探针的活体分子成像,结合荧光透射成像(3DFMT)方法,建立了可进行三维时空动态FRET检测的平台系统,对FRET探针的测量结果(即待测物浓度)进行实时定量的三维重建。结果准确地定量了待测物Ca M浓度,并得到了高质量的3D-FRET分布影像。结论提出FRET探针在活体成像应用领域两个关键问题的解决方案,为基因编码FRET探针向活体分子影像领域的推广奠定了重要的技术基础。     

基于电热型扫描微镜的微型双光子显微镜探头设计

双光子荧光显微镜的微型化对在体脑部神经活动研究和体内组织病理状态检测具有非常重要意义。本文设计了一种基于电热型扫描微镜的微型化双光子荧光显微镜探头,其微镜设计采用反向串联双S型双层材料结构,通过对微镜四个侧面执行器分别施加周期信号,实现了±3.5°的扫描范围;其光学设计是通过单模光纤导入800 nm飞秒激光,激光经微镜扫描,由光学系统聚焦作用于生物样品,激发样品产生荧光,荧光信号通过采集光路汇集并耦合到多模光纤输出,最后通过显微镜的图像采集系统成像。探头尺寸为26 mm(L)× 12 mm(W)× 8 mm(H),重量为5.6 g。利用该显微镜探头,对含有不同尺寸凹槽的微结构进行了成像测试,其结果表明,其扫描成像的范围可以实现150 μm × 150 μm,横向分辨率可达到5 μm;利用该探头对荧光染色小鼠肾脏冷冻切片进行的成像测试,成功得到了双光子二维荧光图像。基于本装置以及进一步的改进,将有助于构建便携式双光子荧光显微镜,置于活体动物头部,在体进行脑部神经活动研究。     

一种水溶性稀土上转换荧光纳米探针及其活体荧光成像研究

目的制备一种水溶性稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋,用于动物活体荧光成像。方法采用三氟乙酸盐热分解法合成油溶性稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋,然后选用柠檬酸或L-半胱氨酸作为修饰剂,置于油-水两相中加热到100℃进行配体交换,离心纯化得到水溶性产物。并对所制备的上转换荧光纳米微粒进行X射线衍射测试（XRD）、X射线光电子能谱分析（XPS）、透射电子显微镜及红外和荧光光谱等表征,确定稀土上转换荧光纳米探针的组成、形态及修饰后该纳米探针的性质。最后将修饰后的水溶性稀土上转换荧光纳米探针作为探针应用于小鼠的活体成像。结果制备的稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋为立方相、平均粒径约21.3 nm的多边形结构,分别在540 nm和660 nm处发出荧光。用柠檬酸或L-半胱氨酸修饰后的稀土上转换荧光纳米探针可以稳定地分散在水中;并且修饰后的稀土上转换荧光纳米探针的粒径、晶相及荧光性质没有发生改变;红外光谱表明,该纳米探针的表面成功引入了修饰剂;该荧光纳米探针用于小鼠活体成像荧光信号清晰可见。结论研制出了一种用柠檬酸或L-半胱氨酸表面修饰的、水溶性稀土上转换荧光纳米探针NaYF4∶Yb3＋/Er3＋,表现出了较好的动物活体成像应用前景。     

Intravascular atherosclerotic imaging with combined fluorescence and optical coherence tomography probe based on a double-clad fiber combiner

We developed a multimodality fluorescence and optical coherence tomography probe based on a double-clad fiber (DCF) combiner. The probe is composed of a DCF combiner, grin lens, and micromotor in the distal end. An integrated swept-source optical coherence tomography and fluorescence intensity imaging system was developed based on the combined probe for the early diagnoses of atherosclerosis. This system is capable of real-time data acquisition and processing as well as image display. For fluorescence imaging, the inflammation of atherosclerosis and necrotic core formed with the annexin V-conjugated Cy5.5 were imaged. Ex vivo imaging of New Zealand white rabbit arteries demonstrated the capability of the combined system.     

量子点探针辅助的近红外荧光成像技术在肿瘤显影中的应用

肿瘤早期检测是精准并高效诊疗癌症的关键因素。荧光成像技术凭借其高灵敏度、高时空分辨率、无电离辐射和无创实时成像等优点,在生物医学领域,尤其在肿瘤检测方面展现出了广泛的应用前景。近红外光穿过生物组织时,受到的吸收和散射较少,因此在生物成像方面体现了高信噪比和强组织穿透能力。在众多荧光探针中,近红外发光的量子点探针因其量子产率高、抗光漂白性强、发射光可调和稳定性良好等特点在荧光成像方面显示出突出的优势。本文基于量子点探针的近红外荧光成像技术在肿瘤显影中的应用,介绍了量子点优异的光学性能,并重点讨论了硫化铅（PbS）和硫化银（Ag2S）近红外发光量子点探针在肿瘤成像方面的研究进展,并对近红外发光量子点探针的应用前景进行了展望。     

Percutaneous optical imaging system to track reporter gene expression from vasculatures in vivo

This study develops a percutaneous optical imaging system for tracking fluorescent reporter gene expression in vasculatures. We build a percutaneous optical imaging system that primarily comprised a 1.5-mm, semi-rigid, two-port optical probe. The performance of the optical probe is first tested in vitro with cell phantoms, and then the feasibility of the percutaneous optical imaging system is validated in vivo in eight femoral artery segments of two pigs. The green fluorescent protein (GFP) gene is locally delivered into four arterial segments, while saline is delivered to the four contralateral arterial segments as controls. The targeted arteries are localized using color Doppler, and thereafter the optical probe is positioned to the target arterial segments under ultrasound guidance. Optical imaging captures are obtained using different exposure times from 10 to 60 s. Subsequently, the GFP- and saline-targeted arteries are harvested for fluorescent microscopy confirmation. The percutaneous optical probe is successfully positioned at a distance approximately 2 mm from the targets in all eight arteries. The in-vivo imaging shows higher average signal intensity in GFP-treated arteries than in saline-treated arteries. This study demonstrates the potential using the percutaneous optical imaging system to monitor, in vivo, reporter gene expression from vasculatures.     

Probe pressure effects on human skin diffuse reflectance and fluorescence spectroscopy measurements

Diffuse reflectance and fluorescence spectroscopy are popular research techniques for noninvasive disease diagnostics. Most systems include an optical fiber probe that transmits and collects optical spectra in contact with the suspected lesion. The purpose of this study is to investigate probe pressure effects on human skin spectroscopic measurements. We conduct an in-vivo experiment on human skin tissue to study the short-term (<2 s) and long-term (>30 s) effects of probe pressure on diffuse reflectance and fluorescence measurements. Short-term light probe pressure (P0<9 mN∕mm2) effects are within 0?±?10% on all physiological properties extracted from diffuse reflectance and fluorescence measurements, and less than 0±5% for diagnostically significant physiological properties. Absorption decreases with site-specific variations due to blood being compressed out of the sampled volume. Reduced scattering coefficient variation is site specific. Intrinsic fluorescence shows a large standard error, although no specific pressure-related trend is observed. Differences in tissue structure and morphology contribute to site-specific probe pressure effects. Therefore, the effects of pressure can be minimized when the pressure is small and applied for a short amount of time; however, long-term and large pressures induce significant distortions in measured spectra.     

Fiber-based combined optical coherence and multiphoton endomicroscopy

A fiber-based multimodal imaging system that combines multiphoton microscopy (MPM) with Fourier domain optical coherence microscopy (OCM) is reported. The system uses a fiber-based femtosecond laser, a fiber coupler, and a double-clad fiber (DCF) device. The fiber laser has a central wavelength of 1.04 μm and bandwidth of 29 nm. Longer excitation wavelength is used to increase penetration depth and increase the excitation efficiency for dyes, such as red fluorescent dyes. A single mode fiber coupler is used to replace the free-space beam splitter and one arm of the coupler is fused with a double-clad fiber device. The MPM and OCM share the same excitation light path in the core of a double-clad fiber, while the OCM and MPM signals were collected by the core and clad of the double-clad fiber, respectively. The performance of the introduced double-clad device is analyzed. The device can confine all the excitation light in the core and has a collection efficiency of 20% for the MPM signal. The efficiency can be further increased by fusing more multimode fibers with the DCF. Simultaneous optical coherence microscopic imaging, second harmonic generation imaging, and two-photon excitation fluorescence imaging are demonstrated in biological samples.     

利用光强分布测量生物组织的光学特性

目的 设计一种利用光强分布测量生物组织光学性质的新方法.方法 利用光纤测定了650 nm连续激光入射到半无限大生物组织模拟液Intralipid- 10％中的光强分布,由漫射强度表达式,通过非线性拟合的方法确定了生物组织的光学特性参数.利用确定的光学参数和光强公式可以了解样品中光强的分布和各点光强的各向异性.结果 通过与现有的结果比较,证明这种方法是可行的.结论 此研究能对液体样品内部的散射性质有更全面的了解,为液体如生物医学用的液体样品的测量和激光诊疗提供有用的信息.     

Near-infrared imaging of fast intrinsic optical responses in visible light-activated amphibian retina

High performance functional imaging is needed for dynamic measurements of neural processing in retina. Emerging techniques for visual prosthesis also require advanced methodology for reliable validation of electromagnetic stimulation of the retina. Imaging of fast intrinsic optical responses associated with neural activation promises a variety of technical advantages over traditional single and multichannel electrophysiological techniques for these purposes, but the application of fast optical signals for neural imaging has been limited by low signal-to-noise ratio and high background light intensity. However, by using an optimized near-infrared probe light and improved optical system, we improve the optical signals substantially, allowing single pass measurements with approximately micron resolution. We image fast intrinsic optical responses with different optical modalities, i.e., bright field, dark field, and cross-polarization, from isolated retina activated by visible light stimulation. At single cell resolution, bright-field imaging discloses the maxima of optical responses approximately 5% dI/I, where dI is the dynamic optical change and I is the baseline light intensity. Dark-field imaging techniques further enhance the sensitivity of optical measurements, and show the maxima of optical responses exceeding 10% dI/I. Cross-polarized imaging provides optical sensitivity similar to dark-field imaging, but different patterns of neural activation are observed.     

Fluorescence lifetime imaging microscopy for brain tumor image-guided surgery

We demonstrate for the first time the application of an endoscopic fluorescence lifetime imaging microscopy (FLIM) system to the intraoperative diagnosis of glioblastoma multiforme (GBM). The clinically compatible FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system with a fiber-bundle (fiber image guide of 0.5 mm diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, and 4 mm field of view) to provide intraoperative access to the surgical field. Experiments conducted in three patients undergoing craniotomy for tumor resection demonstrate that FLIM-derived parameters allow for delineation of tumor from normal cortex. For example, at 460±25-nm wavelength band emission corresponding to NADH/NADPH fluorescence, GBM exhibited a weaker fluorescence intensity (35% less, p-value<0.05) and a longer lifetime τGBM-Amean=1.59±0.24 ns than normal cortex τNC-Amean=1.28±0.04 ns (p-value<0.005). Current results demonstrate the potential use of FLIM as a tool for image-guided surgery of brain tumors.     

Fluorescence bronchoscopy for localization of carcinoma in situ

A fluorescence bronchoscope system has been developed for imaging lung tumors by fluorescence of a previously injected, tumor-specific agent hematoporphyrin derivative. Carcinoma in situ has been localized, but there are too many false positives and negatives. A new system has been implemented which allows rapid switching between viewing of fluorescence, and viewing of the same area under white light illumination as in conventional bronchoscopy. The excitation source is a violet krypton ion laser coupled to a fused quartz fiber light conductor, with a diverging microlens to spread the light uniformly. A third-generation, microchannel plate image intensifier amplifies the weak fluorescence for viewing and video display, recording, and analysis. A movable mirror and periscope bypasses the intensifier for normal color viewing and video display and recording, with the laser shutter closed and the white light shutter open. This facilitates accurate localization, comparison of the color and fluorescence images, and precise sampling during biopsy. The improved system should reduce the false positive rate due to biopsy sampling error, and together with the video analyzer should reduce indeterminate results.     

Integrated micro-endoscopy system for simultaneous fluorescence and optical-resolution photoacoustic imaging

We present a new integrated micro-endoscopy system combining label-free, fiber-based, real-time C-scan optical-resolution photoacoustic microscopy (F-OR-PAM) and a high-resolution fluorescence micro-endoscopy system for visualizing fluorescently labeled cellular components and optically absorbing microvasculature simultaneously. With a diode-pumped 532-nm fiber laser, the F-OR-PAM sub-system is able to reach a resolution of ～7 μm. The fluorescence subsystem, which does not require any mechanical scanning, consists of a 447.5-nm-centered diode laser as the light source, an objective lens, and a CCD camera. Proflavine is used as the fluorescent contrast agent by topical application. The scanning laser and the diode laser light source share the same light path within an optical fiber bundle containing 30,000 individual single-mode fibers. The absorption of proflavine at 532 nm is low, which mitigates absorption bleaching of the contrast agent by the photoacoustic excitation source. We demonstrate imaging in live murine models. The system is able to provide cellular morphology with cellular resolution co-registered with the structural information given by F-OR-PAM. Therefore, the system has the potential to serve as a virtual biopsy technique, helping visualize angiogenesis and the effects of anti-cancer drugs on both cells and the microcirculation, as well as aid in the study of other diseases.