高灵敏度荧光显微镜量化技术及其在光生物学研究中的应用

介绍一种用像增器接收荧光图像的高灵敏度荧光显微镜,相对于普通荧光显微镜的灵敏度提高了４×１０４倍,并用宽量程微光光亮度计对仪器的微弱成像性能进行了实验标定,得到了图像采集数据和图像发光强度的线性数量关系。高灵敏度荧光显微镜在给出细胞荧光图像的同时,可以给出图像上每一像元的发光强度和细胞平均发光强度,仪器对图像细微变化的判断能力远大于人眼直接观察图像。高灵敏度荧光显微镜可应用于研究细胞中荧光物质在细胞生理过程中的分布变化和发光强度变化。使用此仪器已得到了光敏竹红菌甲素（ＨＡ）在Ｈｅｌａ细胞（人体子宫癌细胞）中的分布图像和更为直观的三维显示图形,以及加入ＨＡ后Ｈｅｌａ细胞受到强先照射后的细胞损伤图像。     

光子计数方法探测明亮杆菌的发光图像

本文研制了用生物倒置显微镜和以微通道板为核心的超高灵敏度光电成像系统组成的光子计数显微成像系统,此系统具有高空间放大倍数（１６０×）和极高的光子放大倍数（１０７）,可探测到大小为μｍ量级,发光强度为１００光子／秒的发光图像。应用此系统拍摄到了单个明亮发光杆菌（Ｐｈｏｔｏｂａｃｔｅｒｉｕｍｐｈｏｓｐｈｏｒｅｕｍ）的发光图像,图像显示明亮发光杆菌的大小为２．４×０．６０（μｍ）２,单个细菌的发光强度在１００光子／秒量级。上述实验为明亮杆菌在环境保护中的应用提供了基本参数,也为进一步从细胞水平上研究发光细菌提供了可能。     

不同光波长下大光金鱼花叶片超微弱发光的光子计数统计

用生物超微弱发光探测技术,对植物叶片的超微弱发光透过滤波片进行测量,并用量子光学中的光子计数分布统计方法得到的结果表明,不同波长下,大光金鱼花叶片的光子计数分布不同,但幼叶和老叶的光子计数分布的规律基本相同,而成熟叶片光子计数分布的规律比较紊乱。     

人宫颈癌细胞Hela移植模型肿瘤生长的荧光分析和卡尺测量的比较

目的建立稳定表达绿色荧光蛋白的人宫颈癌细胞系,建立移植瘤模型并比较移植模型肿瘤生长的荧光分析和卡尺测量的优缺点。方法以Lipofectamine 2000介导chickenβ-actin-GFP-NEO转染人宫颈癌细胞Hela,经梯度浓度G418筛选获得稳定表达绿色荧光蛋白的细胞克隆并扩大培养。BALB/cA-nu裸鼠皮下接种1×10^6个发光细胞使其成瘤,利用活体荧光成像系统和游标卡尺观察肿瘤的生长情况。结果获得了稳定表达GFP的人宫颈癌细胞株,将其接种到裸鼠体内可成瘤。活体荧光成像观察发现,1至3周随着肿瘤体积逐渐增大,平均荧光光子数逐渐增加;4周时随着肿瘤出现明显坏死,平均荧光光子数呈现下降趋势,而游标卡尺测量结果显示肿瘤在4至5周仍然不断的增大。结论绿色荧光蛋白能够在人宫颈癌细胞Hela中长期稳定表达,用绿色荧光蛋白标记的人宫颈癌细胞Hela建立的裸鼠肿瘤模型可以为人宫颈癌研究提供理想的实验材料,应用小动物活体成像系统能够客观定量评价活的肿瘤细胞在动物体内的生长情况,而不是肿瘤体积的变化。     

新型数字化高灵敏度荧光显微镜及其在生物学中的应用

尽管普通荧光显微镜已广泛应用于生物医学领域,其性能上还存在一些不足,为了克服它的弱点,借助于像增强器和ＣＣＤ,将倒置生物显微镜改造为数字化高灵敏度荧光显微镜,并增加动态图像获取功能。改进后的荧光显微镜具有以下优点：（１）灵敏度极高,可探测微弱的荧光图像;（２）运用图像融合技术,实现荧光准确定位;（３）适合于观察切片和活细胞;（４）可研究细胞荧光图像的动力学特征;（５）能够给出图像上各点的坐标和对应的发光强度,具有定量显示特点;（６）图像处理软件功能完善,操作方便。利用该荧光显微镜,以吖啶橙为荧光标记物观察正常细胞和凋亡细胞的不同状态,获得良好的实验效果。     

生物系统超弱发光的探测:绿豆、大鼠血液和3T3细胞的发光

本文报道一台用于生物系统微弱发光研究的高灵敏单光子计数系统、能在200—900nm范围测量生物样品的发光强度、光谱和发光动力学.由于光电倍增管以液氮冷却、噪声降至40cps,在99.9%置信度和6小时测量条件下,最小可探测0.3光子/秒、厘米~2的微弱光子流.用该系统研究了萌发绿豆、大鼠血液和体外培养正常和转化3T3细胞的发光.这些样品的发光都含有一个光诱导成份,且以不同的速率衰减、最后达到稳定水平代表了这些生物系统自发的代谢发光.实验发现,转化的3T3细胞的发光强度比正常细胞约低30%.     

大光金鱼花不同叶位叶片超微弱发光的光子计数统计

采用生物超微弱发光探测技术,并用量子光学中的光子计数分布统计方法测量植物叶片超微弱发光的结果表明,大光金鱼花幼叶和老叶的超微弱发光强度较低,光子计数分布与泊松分布基本吻合。     

植物幼苗原发性超弱光子成像的研究

采用光子计数成像系统（PIAS）对植物幼苗萌发过程的超弱发光进行观察。结果表明,自发光子长时间积累可形成二维图象;光子计数和采集图象均可得到植物体的自发发光;通过实验探测到幼苗的根,叶在同一平面图象有不同的发光表现;光子成像系统可客观地比较生物自发超弱发光,为进一步研究超弱发光机理提供实验基础。     

三套荧光显微成像系统在光敏剂亚细胞定位研究中的应用比较

目的：对三套荧光显微成像系统在国产新型光敏剂HMME亚细胞定位研究中的应用特点及适用范围进行了比较与评价。方法：分别应用LSCM、CCD、ICCD荧光显微成像系统,选择特异性细胞器荧光探针Rhodamine-123、DIOC6(3)标记细胞内线粒体和内质网。采用细胞器-细胞荧光强度比值法,对HMME进行单细胞内分布的定性与定量研究。结果：LSCM和CCD成像系统能采集到浓度达到160μg／ml时的HMME的荧光图像,获得荧光探针图像信息显示所标记的细胞内线粒体和内质网平均荧光强度比值(J1/J2值)都明显高于细胞内J1/J2值。而ICCD成像系统只需HMME浓度为5μg/ml,荧光图像特点都呈胞浆中荧光强度较高且分布不均,细胞核区荧光较弱的中空现象。ICCD系统对细胞器探针荧光图像在空间分辨上不理想。结论：LSCM与CCD成像系统限于其探测灵敏度,对于弱荧光性光敏剂,适用于其高孵育浓度条件下的亚细胞定位研究。二者获得的结果相一致：孵育24h,HMME在鼠肺内皮细胞线粒体和内质网有分布而几乎不进入细胞核。ICCD成像系统可不受孵育浓度条件的限制,实现光敏剂极微弱荧光的有效探测,但空间分辨率较低。     

人体超微弱发光图像中的信号检验

用近期研制的具有单光子探测能力的超高灵敏度成像系统获得了人体体表生物超微弱发光的强度数据。为分析图像中的信号检验,二项分布被用于人手不同时间累积发光图像中的信号显著性检验,证实人手存在超微弱生物发光,手指的发光强度在３００～５５０ｐｈｏｔｏｎｓ／ｓ范围内,全手的发光强度在８５０～１２００ｐｈｏｔｏｎｓ／ｓ范围内。     

Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies

Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (iv, ip, sc), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000–300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.     

Low-light imaging technology in the life sciences

Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence. New high-performance low-light level imaging systems have recently become available for the life science. These systems use advances in camera design and digital image processing and are now being used for a wide range of luminescence applications. They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis. This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening. Improvements in imageprocessing hardware and software have increased the usefulness of these systems in the biosciences. Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays. As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections).     

活体生物发光与荧光成像技术在干细胞研究中的应用

活体生物发光与荧光成像技术是近年发展起来的新兴技术,以其操作简便、灵敏度高、创伤性小在生命科学研究中有着较大的优势,目前已被广泛应用于基因标记、细胞凋亡、免疫细胞研究、肿瘤转移等诸多领域,尤其在新兴的干细胞研究方面更是发挥着不可替代的作用。综述了活体生物发光与荧光成像技术的原理、优势、应用范围及发展前景,特别对近年来该技术在胚胎干细胞的肝向分化、人造血干细胞重建小鼠造血系统、神经祖细胞治疗中枢神经系统肿瘤等方面的应用做了详细介绍。     

Photon counting imaging: applications in biomedical research

Photon counting imaging, a technique capable of imaging at the single photon level, is finding applications in biological research and is providing unprecedented views of ultra-low light level phenomena. In combination with the optical microscope, this technique has provided a means of directly visualizing gene expression in single cells, imaging metabolites in tumor tissue and visualizing the chemiluminescence associated with oxidative metabolism in phagocytic cells. At the macroscopic level, it has greatly extended the sensitivity of detection in protein blots and has been applied as an image luminometer to assay microtiter plates. The technique holds great promise for use with fluorescence- and luminescence-based methods in many fields of research.     

Fluorescence and bioluminescence of bacterial luciferase intermediates.

An intermediate in the luciferase-catalyzed bioluminescent oxidation of FMNH2, isolated and purified by chromatography at -20degrees, was postulated to be an oxygenated reduced flavin-luciferase. Maintained and studied at -20 to -30degrees, this material exhibits a relatively weak fluorescence emission peaking about 505 nm when excited at 370 nm. It may comprise more than one species. Upon continued exposure to light at 370 nm, the intensity of this fluorescence increases, often by a factor of 5 or more, and its emission spectrum is blue shifted to a maximum at about 485 nm. Upon warming its fluorescence is lost and the fluorescence of flaving mononucleotide appears. If warming is carried out in the presence of a long chain aldehyde, bioluminescence occurs, with the appearance of a similar amount of flavine fluorescence. The bioluminescence yield is about the same with irradiated and nonirradiated samples. The bioluminescence emission spectrum corresponds exactly to the fluorescence emission spectrum of the intermediate formed by irradiation, implicating the latter as being structurally close to the emitting species in bioluminescence.     

Looking and listening to light: the evolution of whole-body photonic imaging

Optical imaging of live animals has grown into an important tool in biomedical research as advances in photonic technology and reporter strategies have led to widespread exploration of biological processes in vivo. Although much attention has been paid to microscopy, macroscopic imaging has allowed small-animal imaging with larger fields of view (from several millimeters to several centimeters depending on implementation). Photographic methods have been the mainstay for fluorescence and bioluminescence macroscopy in whole animals, but emphasis is shifting to photonic methods that use tomographic principles to noninvasively image optical contrast at depths of several millimeters to centimeters with high sensitivity and sub-millimeter to millimeter resolution. Recent theoretical and instrumentation advances allow the use of large data sets and multiple projections and offer practical systems for quantitative, three-dimensional whole-body images. For photonic imaging to fully realize its potential, however, further progress will be needed in refining optical inversion methods and data acquisition techniques.     

Bioluminescent system for dynamic imaging of cell and animal behavior

The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.     

一种新的基于蒙特卡罗模拟和光子传输模型的图像分割算法

本文以蒙特卡罗模拟方法为基础,结合组织光学的光子传输模型,提出了一种新的图像分割算法,该算法将复杂的图像分割问题简化为大量简单的光子传输随机实验,通过分析传输规律来获取目标区域.在随后的实验中,结合细胞核提取这一问题建立了一个简单的光学传输模型,并依据此模型分别对人造图和实际图进行了分割.人造图的分割结果表明了该算法的可行性,说明了该算法的一些优点;而实际图的分割结果则反映了该算法的不足之处,文章针对其中存在的问题和算法待改进之处进行了分析.     

Photomovement of the red alga Porphyridium cruentum (Ag.) Naegeli

Phototaxis of the unicellular red alga Porphyridium cruentum was studied by staining the slime tracks of individual cells as well as with the aid of a population method. Because of the increased straightness of the movement the mean linear velocity of a unilaterally illuminated population exceeds considerably that of an only photokinetically stimulated one. In white light the phototactic reaction is saturated already at 100 lx. The zero threshold lies at about 1 lx. Spectral sensitivity curves of phototaxis obtained at high photon fluence rates (>=10^–11 mol cm^–2 s^–1) display two main peaks which shift against each other at intermediate irradiances and, finally, form a single maximum in the blue range (443 nm) at low photon fluence rates (10^–12 and 10^–13 mol cm^–2 s^–1). Photon fluence rate-response curves reveal that supraoptimal irradiances decrease the phototactic reaction, especially in the range of the highest sensitivity of the cells. The action spectrum of phototaxis was calculated on the basis of the photon fluence rate-response curves. It shows a maximum at 443 nm and shoulder at 416 nm and between 467 and 477 nm. Wavelengths longer than 540 nm are phototactically inactive even at very high irradiances (25 W m^–2). Thus, this is the first phototactic action spectrum of a biliprotein-containing organism which does not indicate the participation of biliproteins in the absorption of phototactically active light. DCMU and potassium iodide have no specific effects on phototaxis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea