科技消息

以激光为光源的细胞荧光分析装置此装置以氦镉激光器(422nm)为光源,荧光显微镜与荧光光谱仪联用。在砷化镓光电倍增管前装有双光栅单色器,并与台式计算机相连。此种分析装置适用于细胞生物学的研究,可以定量定位的测出一个细胞或细胞中某一部位的荧光强度,如细胞核经Feulgen-Schiff染色后,用激光光源的荧光显微镜测定,其荧光强度要比以汞灯为光源的高10—100倍,大大提高了核酸检出灵敏度,因此,可精确地分析比较细胞间的或细胞各部位间的荧光强度变化,也可准确和快速地     

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%.     

声光可调谐滤波器的细胞生物学研究应用

波长选择在荧光光谱仪和显微镜等光学应用中发挥了至关重要的作用。声光可调谐滤波器(AOTF)作为一种电光器件可实现多光源入射波长、功率的同时调制。在声光可调谐滤波器中,压电换能器结合于二氧化碲或石英晶体产生高频声波,改变晶体折射率形成周期性分布。该现象在晶体中生成衍射光栅,使以布拉格角正交入射的光束被高效衍射至一阶光束。当改变施加到晶体的信号频率时将改变折射率变化周期,因此,衍射光的波长随之改变。同时,衍射光强度由施加到晶体的信号振幅决定。本文从声光可调谐滤波器原理和特点出发,总结了声光可调谐滤波器在细胞生物学研究系统中的应用模型。得益于作用时间短、波长分辨率高、无振动部件等特性,声光可调谐滤波器提升了多波长光源功率调制能力,使细胞计数系统具备了细胞高光谱成像能力。所以不仅限于传统细胞生物学研究,包含声光可调谐滤波器件的系统还将在多参数高内涵成像分析、扫描荧光显微术、药物毒理研究等领域成为有力的研究工具。     

新型快速显微多道分光光度系统及其应用

介绍一种新型快速显微多道分光光度系统。利用倒置显微镜、衍射光栅、线阵CCD等构成的显微多道分光光度系统,可对微区样品在350nm-800nm（波长分辨率为0．2nm)光谱波长范围内进行快速光谱检测,最快检测时间为1ms。利用该系统进行的人血红细胞内血红蛋白测量分析表明,该具有高灵敏度、快速、无扰、在位、多光谱显微测量的特点,在生命科学研究中动态监测细胞生命过程的变化。     

利用超快时间分辨技术对光系统Ⅱ核心天线CP43和CP47的动力学荧光光谱的分析

采用超快时间分辨荧光光谱装置对光系统Ⅱ核心天线CP43和CP47进行了研究 ,并在 5 14.5nm激光激发下获得了它们的动力学荧光光谱。CP43和CP47的荧光光谱范围分别为 6 40～ 780nm和 6 30～ 775nm ,并且它们分别在约 6 80nm和 6 91nm处有最大峰 ,在这两个峰值处的荧光寿命分别约为 3.5 4ns和 3.2 2ns。通过理论计算认为在CP43和CP47中 ,叶绿素a的荧光发射效率分别约为 38.3%和 40 .6 %。讨论了类胡萝卜素到叶绿素a分子的能量传递 ,认为在CP43和CP47中 ,类胡萝卜素到叶绿素a分子的能量传递时间常数分别为 9.6× 10 11s-1和 1.3× 10 12s-1,能量传递效率分别为 47.5 %和 6 6 .5 % ,并且估计在这两种核心天线中 ,类胡萝卜素分子和叶绿素a分子的外周间距分别约为 0 .110nm和 0 .0 85nm。     

去镁叶绿素置换细菌去镁叶绿素对紫细菌反应中心皮秒荧光的影响

选择597 nm作为激发波长,探测范围为600～900 nm的荧光特性,分析了天然反应中心和两种去镁叶绿素置换的紫细菌反应中心的荧光发射光谱.借助细菌叶绿素、细菌去镁叶绿素和植物去镁叶绿素的荧光光谱,对相关组分进行了归类.实验结果表明选择性地置换细菌去镁叶绿素影响了荧光光谱的组成.在天然反应中心、BpheB置换的反应中心和BpheA,B置换的反应中心中可分别解析到4、3和2个荧光发射组分.研究肯定荧光发射组分与去镁叶绿素的结合存在对应关系.实验还分别在686.4、674.1和681.1 nm处测定了不同反应中心内的原初电子供体P的激发态通过荧光衰减的过程,观测到衰减动力学上的差异.说明去镁叶绿素置换影响了细菌反应中心内激发光能传递和原初光化学反应过程.     

基于光学相干检测的非接触全光学光声显微镜

报道了一种非接触、宽频带、联合微型激光器和低相干迈克尔逊干涉仪的全光学光声显微镜(BD-AOPAM)、光学相干层析系统(OCT)的硬件用于光声信号的检测。目前全光学光声显微镜可检测到的带宽为67 MHz,用碳纤维测得系统的横向分辨率可以达到10.8μm。进一步的,利用包埋头发丝的模拟样品和在体小鼠耳朵血管来验证系统的成像能力。实验结果表明,这种全光学光声显微镜可以在体的实现组织高分辨率的成像,有望成为一种便携式非接触的光声显微镜应用于生物医学当中。     

微秒级动力学光谱型闪光光解装置的研制和某些光解瞬态产物的观察

本文介绍了最近在中国科学院生物物理所建成并投入使用的微秒级动力学光谱型闪光光解装置。该装置的时间分辨率约5微秒,一次闪光消耗的能量为400～600焦耳,瞬态光吸收产物的探测灵敏度达到7×10~(-4)O.D.。在该装置上对水合电子和双卤素阴离子自由基的形成和衰变作了观察,水合电子的最大吸收在680nm处,·Cl_2~-、·Br_2~-和I_2~-的瞬态吸收最大值分别在340nm、355nm和380nm波长处。本文还对这些瞬态产物的衰变动力学作了初步探讨。     

诊断恶性肿瘤的新仪器——固有荧光诊断仪问世

上海市长宁区中心医院和上海医疗器械研究所等单位,研制成检测恶性肿瘤的固有荧光诊断仪。这一居国际先进水平的研究成果最近在上海通过鉴定。整套装置由氮分子激光器、透镜、石英光导纤维、固有荧光光谱测定系统及报警装置等组成。当激光器发出的激光束通过透镜、石英光导纤维照射到所需检测部位时,如呈现深绿色即可排除癌症;如出现桔红色即为癌症。除了肉眼直接观察固有荧光色彩外,还可通过普通多股光导纤维传入自动分色系统,经过一系列电路处理,一秒钟即能报警、显示、记录并可得到一幅固有荧光光谱曲线图,从曲线上即可判明是否有癌。     

去镁叶绿素置换细菌去镁叶绿素对紫细菌反应中心皮秒荧光的影响

选择５９７ｎｍ作为激发波长,探测范围为６００～９００ｎｍ的荧光特性,分析了天然反应中心和两种去镁叶绿素置换的紫细菌反应中心的荧光发射光谱。借助细菌叶绿素、细菌去镁叶绿素和植物去镁叶绿素的荧光光谱,对相关组分进行了归类。实验结果表明,选择性地置换细菌去镁叶绿素影响了荧光光谱的组成。在天然反应中心,ＢｐｈｅＢ置换的反应中心和ＢｐｈｅＡ,Ｂ置换的反应中心中可分别解析到４、３和２个荧光发射组分。研究肯定荧     

Quality assessment of confocal microscopy slide based systems: performance.

BACKGROUND: All fluorescence slide-based cytometry detections systems basically include the following components: (1) an excitation light source, (2) intermediate optics, and (3) a detection device consisting of a CCD camera or a PMT. The optical principles employed is slide-based systems are similar to those of confocal microscopes (CLSM). METHODS: The following tests evaluated confocal equipment performance: dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, PMT reliability, and system noise. RESULTS: Quality assurance tests provide a basis to determine if the equipment is operating correctly. Laser power, PMTs function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance and laser stability were tested colocalization of UV and visible peaks of a bead should be less than 210 nm. Interference contrast optics decrease fluorescence resolution. CONCLUSIONS: QA tests that assess CLSM system performance are also applicable to other slide-based systems. By utilization this type of testing approach, the subjective nature of assessing the CLSM may be eliminated. These tests serve as guidelines for other investigators to ensure that their machines are providing data that is accurate with the necessary resolution, sensitivity and precision.     

The characterization of plant species using first‐derivative fluorescence spectra

Plants are one of the most important parts of the ecological system and demand a reliable method for accurate classification. In this study, the first‐derivative fluorescence spectral curves (FDFSCs) based on laser‐induced fluorescence technology were proposed for the characterization of plant species. The measurement system is mainly composed of a spectrometer, an excitation light source (the two excitation wavelengths are 460 and 556 nm, respectively), and an intensified charge‐coupled device camera. FDFSCs were calculated from the deviation between the fluorescence values at each wavelength, plus and minus one band, divided by the wavelength range. Principal component analysis was utilized to analyze the FDFSCs by extracting the main attributes and reducing the dimensionality of variables. A support vector machine was used to evaluate FDFSC performance for the identification of plant species. Plant species that are difficult to distinguished by the naked eye, can be identified effectively using the proposed FDFSCs. For the 556 nm and 460 nm excitation wavelengths, the overall identification rates of the six plant species evaluated were 93.3% and 91.7%, respectively. Experimental results demonstrated that the combination of the FDFSCs with multivariate analysis could provide a simple and reliable method for the characterization of plant species.     

Picosecond transient absorption study of photodissociated carboxy hemoglobin and myoglobin.

The optical transient absorption spectra at 30 ps and 6.5 ns after photolysis are compared for both carboxy hemoglobin (HbCO) and carboxy myoglobin (MbCO). Both 355- and 532-nm excitation pulses were used. In all cases the shapes of the optical difference spectra thus generated are stationary over the complete time-scale studied. The photolysis spectra for MbCO are not significantly different from the equilibrium difference spectra generated on the same picosecond spectrometer when measured to an accuracy of +/- 0.5 nm. In addition, spectral parameters for delegated HbCO generated on the same spectrometer but detected by two different techniques, either by a Vidicon detector or point by point with photomultiplier tubes, are reported; the results are different from some of the previously reported picosecond experiments.     

A subnanosecond resolving spectrofluorimeter for the analysis of protein fluorescence kinetics

A spectrofluorometer is described consisting of an excitation source, optics, detector and time resolving electronics. The excitation source consists of a mode-locked Ar ion laser, synchronously pumps a dye laser, followed by a frequency doubling device. The repetition frequency of the U.V. pulses (FWHM some ps) has been reduced by an extra-cavity electro-optical modulator. Provisions have been made in the optical configuration to determine both time-resolved fluorescence spectra and fluorescence anisotropy decay curves. The commercially avialable electronics have been optimized for maximum time resolution. The spectral output of the excitation source is confined between 280 and 310 nm, which encompasses the region for eliciting protein fluorescence. The performance of the complete system has been tested with single lifetime standards line p-terphenyl in cyclohexane or with $\text{N-}\text{acetyl}\text{-}\text{L}\text{-}\text{tryptophanamide}$ in pH 7.5 buffer. Serum albumins from human and bovine sources have been employed as examples for time resolved fluorescence spectra and for the demonstration of anisotropy decay curves. Using these methods protein dynamics in the (sub)nanosecond time region can be directly explored.     

One- and two-photon-induced fluorescence from recombinant green fluorescent protein

The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser. Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon. The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra. A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation.     

Self-phase-modulated femtosecond laser source at 1603 nm and its application to deep-brain 3-photon microscopy in vivo

3-photon microscopy (3PM) excited at the 1700 nm window enables deep-tissue imaging in vivo, especially in brain. PC rod soliton source has previously been exclusively used as the excitation source, which is rather costly and difficult to align. Here we demonstrate a novel nonlinear optical technique to build femtosecond laser source at the 1700 nm window, based on self-phase modulation (SPM) in a short span of large-mode-area fiber. The spectral broadening experienced by the pump pulse leads to the generation of a red-shifted sidelobe at 1603 nm. After spectral filtering, this sidelobe corresponds to 170-fs, 167-nJ pulses at 1603 nm. Using this SPM source, we further demonstrate deep-brain 3 PM to a depth of 1500 μm below the mouse brain surface in vivo. Our SPM femtosecond laser source thus provides a cost effective and easy-to-align alternative excitation source to the PC rod soliton source.     

Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra

To conduct rapid microscope observations with the excitation spectral measurement for photosynthetic organisms, a wavelength-dispersive line-focus microscope was developed. In the developed system, fluorescence signals at multiple positions on a sample excited with different wavelengths can be detected as a two-dimensional image on the EMCCD camera at the same time. Using the developed system, one can obtain excitation spectra at every pixel over the excitation wavelength range from 635 to 695 nm, which covers the full range of the Q_y bands of both chlorophyll-a and chlorophyll-b. Recording the reference laser spectra at the same time ensures robust measurement against the moderate spectral fluctuation in the excitation laser. Using an objective lens with a numerical aperture of 0.9, the lateral and axial resolutions of 0.56 and 1.08 μm, respectively, were achieved. The theoretically limited and experimentally estimated spectral resolutions of the excitation spectral measurement were 0.86 and 1.3 nm, respectively. The validity of the system was demonstrated by measuring fluorescent beads and single cells of a model alga, Chlamydomonas reinhardtii. Intrachloroplast inhomogeneity in the relative intensity of the chlorophyll-b band could be visualized in Chlamydomonas cells. The inhomogeneity reflects the intrachloroplast variation in the local peripheral antenna size.     

Two-photon excitation fluorescence spectrum of the light-harvesting complex LH2 from Chromatium minutissimum within 650-745 nm range is determined by two-photon absorption of bacteriochlorophyll rather than of carotenoids

Two-photon fluorescence excitation spectra of the peripheral light-harvesting complex LH2 from the purple photosynthetic bacterium Chromatium minutissimum were examined within the expected spectral range of the optically forbidden S1 singlet state of carotenoids. LH2 preparations isolated from wild-type and carotenoid-depleted cells were used. 100-fs laser pulses in the range of 1300-1490 nm with an energy of 7-9 mW (corresponding to one-photon absorption between 650 and 745 nm) were used for two-photon fluorescence excitation. It was shown that two-photon fluorescence excitation spectra of LH2 complex from wild and carotenoid-depleted cells are very similar to each other and to the two-photon fluorescence excitation spectrum of bacteriochlorophyll a in acetone. It was concluded that direct two-photon excitation of bacteriochlorophyll a determines the fluorescence of both samples within the 650-745 nm spectral range.     

Practical Confocal Microscopy and the Evaluation of System Performance

The laser scanning confocal microscope has enormous potential in many fields of biology. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by the user's laboratory. To achieve better performance from the equipment, it is necessary to run a series of tests to ensure that the optical machine is functioning properly. We have devised these methods on the Leica TCS-SP and TCS-4D systems. Tests measuring field illumination, lens clarity, laser power output, dichroic functioning, spectral alignment, axial resolution, laser power stability, machine performance, and system noise were derived to test the Leica laser scanning confocal microscopy system. These tests should be applicable to other manufacturers' systems as well. The relationship between photomultiplier tube (PMT) voltage, laser power, and averaging using a 10-microm-diameter test bead has shown that the noise (coefficient of variation of bead intensity, CV) in an image increases as the PMT increases. Therefore increasing the PMT setting results in increased noise. For ideal image quality, it appears that it is better to decrease the PMT setting and increase laser power, as noise generated by high PMT settings will reduce the image quality far more than the bleaching caused by higher laser power. Averaging can be used to improve the image at high PMT values, provided the sample is not bleached by repeated passes of the laser.     

Comparative Evaluation Of Raman Spectroscopy At Different Wavelengths For Extremophile Exemplars

Raman spectra have been obtained for extremophiles from several geological environments; selected examples have been taken from hot and cold deserts comprising psychrophiles, thermophiles and halophiles. The purpose of this study is the assessment of the effect of the wavelength of the laser excitation on the ability to determine unique information from the Raman spectra about the specificity of detection of biomolecules produced as a result of the survival strategies adopted by organisms in extreme terrestrial environments. It was concluded that whereas FT-Raman spectroscopy at 1064 nm gave good quality results the time required to record the data was relatively large compared with other wavelengths of excitation but that better access to the CH stretching region for organic molecules was given. Shorter wavelength excitation of biomolecules in the blue-green regions of the visible spectrum using a conventional dispersive spectrometer was more rapid but very dependent upon the type of chemical compound being studied; most relevant biomolecules fluoresced at these wavelengths but carotenoids exhibited a resonance effect which resulted in an improved detection capability. Minerals and geological materials, in contrast, were best studied at these visible wavelengths. In general, the best compromise system for the excitation of the Raman spectra of both geological and biological materials was provided using a 785 nm laser coupled with a dispersive spectrometer, especially for accessing the 1800–200 cm⁻¹ wavenumber shift region where much of the definitive analytical information resides. This work will have conclusions relevant to the use of miniaturised Raman spectrometers for the detection of biomolecules in extraterrestrial planetary exploration.