激光、激光技术 激光应用 激光生物学

Q631 2006032056双光子激发荧光成像技术对小鼠植入前胚胎的实时观测=Application of two-photon excitation fluorescence i ma-ging to real-ti me investigation of mouse prei mplantationembryo[刊,中]/程兵(清华大学原子分子纳米科学教育部重点实验室.北京(100084)) ,林丹樱…∥光谱学与光谱分析.—2006 ,26(2) .—193-197选取锁模飞秒掺钛蓝宝石(Ti-sapphire)激光器输出的730 nm激光,分别激发Hoechst33342 ,Fluo-4 ,PI和Indo-1四种常用生物荧光染料,分别利用(455±15) nm,(540±15) nm,(580±16) nm和(500±15) nm四种滤光片获得…     

有机染料CSPI的双光子吸收和上转换荧光性质

报道了一种有机染料Trans 4 [p carbazol 9 ylstyryl] ] N methylpyridiniumiodide的线性和非线性光学性质。当用 830nm皮秒激光激发时 ,可以得到强烈的黄绿色上转换荧光 ,中心波长在 538nm附近。该染料的双光子荧光寿命为 95 .6ps。测试了该染料在 72 0～ 1 0 50nm的非线性透过率 ,并求出了该染料在上述波长时的双光子吸收截面。双光子吸收最强在 80 0nm ,吸收截面为 9.2× 1 0 - 48cm4 ·s/ photon。     

双光子激发时间分辨荧光光谱测量技术

建立了一台基于高重复频率扫描相机的双光子激发时间分辨荧光光谱测量系统，能够同时测量样品的荧光光谱和寿命. 该系统的时间分辨率为6.5—200ps，光谱分辨率为1—3nm，能够实现快速数据采集以及可靠和可重复的寿命和光谱测量. 利用标准荧光染料(若丹明6G、香豆素314)及其混合溶液对该系统进行了测试，所得到的荧光光谱分布和寿命值与文献报道一致. 实验结果表明，该系统能有效区分多组分荧光团. 这为鉴别多荧光团或多组分生物组织提供了一种独特的对比方法，可用于多光谱分辨荧光寿命成像和荧光共振能量转移成像等方面. 关键词： 荧光寿命 荧光光谱 双光子激发 高重复频率扫描相机     

激光波长对荧光激发谱分布影响研究

激光器是DNA测序仪中的重要部件,寻求合适波长的激光器可有效提高DNA测序仪性能。分析了DNA检测所用的四种荧光染料FAM、JOE、TAMRA和ROX的激发谱和发射谱,设计实验技术方案利用488nm、505nm和515nm三种不同波长的激光器分别对其进行激发。在相同的积分时间和相同的功率下,488nm激光器对于荧光染料ROX的激发强度太低,515nm激光器对于荧光染料FAM的激发强度太低,而505nm激光器对于四种荧光染料的激发强度均比较强。实验结果表明在三种不同波长中,505nm激光器对四种荧光染料的激发效果最佳,可以替代目前大多数DNA测序仪中的氩离子激光器。     

长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。     

基于自相位调制光谱选择驱动的无标记自发荧光多倍频显微镜系统

由飞秒激光驱动的非线性光学显微镜技术在医学组织成像中具有很多独特的优势,包括多种成像模态、高对比度、高分辨率和无标记的深层光学切片能力等.由于缺乏波长可灵活调谐的飞秒激发光源,导致在多模态成像的同时难以兼顾每种模态的对比度,从而制约了非线性光学显微镜在医学诊断中的广泛应用.本文采用基于自相位调制光谱选择的光纤激光光源,获得了中心波长在990—1110 nm范围内可调谐的高能量飞秒脉冲,并用于驱动非线性光学显微镜.采用990 nm的飞秒脉冲,通过双光子激发荧光和二倍频对胃组织成像,进一步结合图像拼接技术成功获得了胃组织的双模态大视场图像;利用1110 nm的飞秒脉冲,实现了无标记自发荧光多倍频显微镜技术,同时高效激发了胃组织的双光子激发荧光、三光子激发荧光、二倍频和三倍频信号,获得了胃组织的多模态图像.     

双光子共振四波差频产生的真空紫外激光特性研究

介绍以惰性气体Xe为非线性介质、利用双光子共振四波混频差频方法产生的可调谐真空紫外激光(VUV)的特性研究 .实验中 ,传播方向和空间均相互重合的两束脉冲染料激光经透镜聚焦于Xe气混频池内 ,其中一束激光的波长 (2 4 9.6 2 6nm)对应于Xe原子 6P[1/2 ,0 ]←← 5P的双光子共振跃迁 ,通过调谐另一束激光的波长 ,产生在 15 1～ 171nm可连续调谐的VUV激光 .经估算 ,产生的VUV激光的强度约为每脉冲约 0 .2 μJ ,转换效率约为 0 .1% .通过测量超声射流冷却下CO分子的A1Π←X1Σ+ (0 ,0 )带的转动分辨激光诱导荧光 (LIF)光谱 ,确定出VUV激光的线宽为 0 .3cm-1.此外 ,还对VUV激光强度与惰性气体压力、染料激光强度等依赖关系进行了研究 .     

双氰基荧光染料的合成、光学性质及其生物成像

设计合成了一种A-π-D-π-A型的双光子荧光染料3,6-双(4-乙烯基苯腈)-9-乙基咔唑,测试了其在二氯甲烷(DCM)、乙酸乙酯(EA)、乙醇(Et OH)、乙腈(ACN)、二甲亚砜(DMSO)和磷酸缓冲盐溶液(PBS)等不同溶剂中的紫外吸收光谱、单光子及双光子荧光光谱。化合物3,6-双(4-乙烯基苯腈)-9-乙基咔唑在紫外吸收光谱中存在两个相似的特征吸收带并呈现出复杂的溶剂化效应,在DMSO中具有最大荧光量子产率(86.02%),其相应的活性吸收截面为12.56 GM。在双光子荧光成像方面,染料分子具有优良的细胞膜通透性并且在双光子荧光显微镜下呈现出明亮的绿色荧光,表现出较好的双光子荧光成像性能。这些数据表明,化合物3,6-双(4-乙烯基苯腈)-9-乙基咔唑可用作一种较为理想的双光子荧光标记染料。     

基于激光共焦扫描术的亚心形扁藻显微图像与光谱

采用激光共聚焦扫描显微技术,针对亚心形扁藻开展了研究.从获得的488 nm Ar+激光单光子激发的亚心形扁藻自体荧光光谱与图像,可知细胞内有一杯状叶绿体物质,其荧光峰值为682 nm,对应叶绿体发出的红色荧光.在单通道模式下,获得800 nm fs激光双光子激发的扁藻自体荧光光谱与图像,可知每个杯状叶绿体的内部有一个自体荧光更强的圆形物质.在双通道模式下,可分别获得小圆形物质的自体荧光图像,杯状叶绿体自体荧光图像,以及两个通道图像的叠加.进一步获得了双光子藻细胞荧光图的6个主要的荧光峰.采用单光子激光激发可获得亚心形扁藻叶绿体自体荧光图像及其荧光光谱,而双光子激光激发荧光光谱的多通道以及Lambda模式下采集光谱信号与图像,不仅可观察到亚心形扁藻的内部形态结构,还可能从双光子激发荧光图中研究分析亚心形扁藻生化物质的存在,灵敏度较单光子激发高.激光扫描共聚焦显微技术,特别是双光子荧光与图像技术可为海藻的检测与研究提供一种快速、实时、有效、简便的方法.     

脉冲压缩在双光子荧光显微成像中的应用研究

本刊讯自20世纪90年代末,双光子荧光显微成像技术已经逐步成为利用光学手段研究生物组织和细胞的重要工具。由于双光子激发属于非线性光学过程,     

Integrated multimodal endomicroscopy platform for simultaneous en face optical coherence and two-photon fluorescence imaging

We report an all-fiber-optic scanning, multimodal endomicroscope capable of simultaneous optical coherence tomography (OCT) and two-photon fluorescence (TPF) imaging. Both imaging modalities share the same miniature fiber-optic scanning endomicroscope, which consists of a double-clad fiber with a core operating in single mode at both the OCT (1310 nm) and two-photon excitation (1550 nm) wavelengths, a piezoelectric two-dimensional fiber-optic beam scanner, and a miniature aspherical compound lens suitable for simultaneous acquisition of en face OCT and TPF images. A fiber-optic wavelength division multiplexer was employed in the integrated platform to combine the low coherence OCT light source and the femtosecond two-photon excitation laser into the same optical path. Preliminary imaging results of cell cultures and mouse tissue ex vivo demonstrate the feasibility of simultaneous real-time OCT and TPF imaging in a scanning endomicroscopy setting for the first time.     

Two-Color Two-Photon Fluorescence Laser Scanning Microscopy

We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.     

Fluorescence Study of DNA–Dye Complexes Using One-Photon and Two-Photon Picosecond Excitation

Preliminary results of investigation of one-photon- and two-photon-induced fluorescence of acridine orange (AO), epirubicin (ER), hypericin (HYP), and ethidium bromide (EB) in complexes with DNA are presented. A spectrofluorometer based on a picosecond Nd:YAG laser was used for investigations of two-photon (1064-nm, 1-mJ, 40-ps) and one-photon (532- and 355-nm) dye excitation. The spectra of two-photon-induced fluorescence of dyes and their complexes with DNA as well as the kinetics of dyes' fluorescence intensification during their interactions with DNA in dependence on the biomacromolecule concentration were obtained. The intensities of AO, HYP, and EB fluorescence were increased 2.4, 3.2, and 8 times, respectively, after binding with DNA at two-photon excitation, while at one-photon excitation the corresponding values were 2.5, 3.7, and 10 times. The difference in fluorescence enhancement during DNA–dye complex formation at linear and nonlinear excitation may possibly be associated with the fact that the cross sections of one-photon and two-photon absorption, in general, change unequally during the binding of dyes to organic molecules and bathocromic shift of the electronic transitions. It was shown that the peak of AO fluorescence shifted to a longer wavelength on 10 nm after two-photon excitation at 1064 nm in comparison with one-photon excitation at 532 nm.     

小鼠卵母细胞染色体三维双光子荧光图像的轴向衰减

双光子荧光显微镜作为一种高分辨光学仪器,已经被广泛应用于生物样品的非侵入式三维光学成像中。相比共聚焦显微镜,双光子荧光显微镜拥有更深的探测深度。然而,即便如此,在对较厚的生物样品进行非侵入式光学三维成像时,样品的成像质量也往往会随着探测深度的增加而下降。在临床和生物学领域对研究母性遗传起重要作用的小鼠卵母细胞拥有较大的直径(80～100 μm),吸收和散射效应较为明显。本文研究小鼠卵母细胞染色体的三维双光子荧光图像随探测深度增加图像质量的衰减程度。通过对所得图像进行轴向衰减矫正,利用体积作为参数,将矫正前后小鼠卵母细胞内染色体三维双光子荧光图像进行对比。结果表明,由于吸收和散射效应,卵母细胞存在较严重的光学轴向衰减问题,因此,对用双光子荧光三维成像手段获得的小鼠卵母细胞图像进行衰减矫正是有必要的。这为进一步精确定量的研究卵母细胞内染色体的三维构像打下良好的基础。     

Increasing the imaging depth of coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.     

Rb（7^2D）态与基态K原子的碰撞能量转移

Ｋ－Ｒｂ混合蒸汽中，使用Ｒｂ光谱灯和染料激光器，将基态Ｒｂ原子二步激发到７＾２Ｄ态用荧光法测量了过程Ｒｂ（７＾２Ｄ）＋Ｋ（４Ｓ）→Ｒｂ（５Ｓ）＋Ｋ（７Ｓ，５Ｄ），的碰撞转移截面，Ｋ７Ｓ，５Ｄ对Ｒｂ７＾２Ｄ的荧光比中，含Ｋ７Ｓ＾←→５Ｄ碰撞转移的影响，第二个实验可以消除这个影响，利用Ｋ光谱灯和染料激光器产生Ｋ７Ｓ或５Ｄ态，探测Ｋ７Ｓ（５Ｄ）对５Ｄ（７Ｓ）的荧光比，Ｒｂ７Ｄ→Ｋ７Ｓ，５Ｄ碰撞转移截面（     

Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation

We examined the steady-state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for OPE- and TPE. The anisotropy decay measurements of Hoechst 33342 in ethanol revealed the same correlation times for TPE as observed for OPE. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for TPE than for OPE. The anisotropy spectra of Hoechst 33342 were examined in glycerol at ?20°C, revealing limiting values close to the theoretical limits for OPE (0.4) and TPE (0.57). The steady-state anisotropy for OPE decreases in the shorter-wavelength region (R6G dye laser, 280–315 nm), but the two-photon anisotropy for 560 to 630-nm excitation remains as high as in the long-wavelength region (690–770 nm). This result suggests that one-photon absorption is due to two electronic, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm. Our demonstration of these favorable two-photon properties for Hoechst 33342, and the high photostability of the dye reported by other laboratories, suggests that this dye will be valuable for time-resolved studies of DNA with TPE and for two-photon fluorescence microscopy.     

Efficient two-photon sensitized luminescence of europium （Ⅲ） complex based on hypersensitive transitions

Red frequency-upconversion fluorescence emission is observed in europium(Ⅲ) complex with encapsulating polybenzimidazole tripodal ligands, pumped with 930- and 1070-nm picosecond laser pulses. The lumines- cence of transition 5D0 →7F2 (612 nm) is induced by two-photon absorption of hypersensitive transitions 7F0 →5D2 (465 nm) and 7F1 →5D1 (535 nm). Analysis results suggest that the two-photon excitation strength of these hypersensitive transitions is increased dramatically owing to the C3 symmetry of the coordination field.     

Two-color two-photon 4Pi fluorescence microscopy

In 4Pi fluorescence microscopy the point-spread function is composed of a strong central lobe accompanied by interference sidelobes that produce artifacts in the image. We propose to combine two-color two-photon fluorescence microscopy and 4Pi fluorescence microscopy to overcome this sidelobe problem. Simulation results show that a single sharp fluorescence spot can be produced by use of two excitation wavelengths of 400 and 800 nm and detected at 350-nm emission wavelength.     

双光子吸收材料二苯乙烯衍生物的合成及光谱性质

设计、合成并用红外光谱、1H NMR、元素分析表征了三种用于双光子吸收材料的二苯乙烯衍生物,4,4′-双(二苯氨基-反式-苯乙烯基)联苯(BPSBP),4,4′-双(二乙氨基-反式-苯乙烯基)联苯(BESBP)和4,4′-双(9-咔唑基-反式-苯乙烯基)联苯(BCSBP)。实验结果表明三者最强的单光子吸收出现在350～400 nm之间,且单光子吸收和荧光光谱中表现出明显的溶剂化显色效应,揭示了分子内对称电荷转移的本质,双光子荧光光谱则揭示了单光子和双光子吸收具有相同的发射机理。利用双光子上转换荧光法测试发现,三种双光子吸收材料在800 nm飞秒激光的激发下具有较大的吸收截面,分别为892,617和483 GM,这表明在双光子领域有潜在的应用价值。