记新一代微型化高分辨率双光子显微镜的诞生——程和平院士专访

正在生物医学领域,成像技术是最重要的技术手段之一,它让研究者能够观察到组织和细胞内正在发生的过程,为各项研究提供直接而确切的证据.传统的单光子荧光显微镜用单个光子将荧光分子激发到激发态,进而产生可被观测的荧光,而发明于1990年的双光子显微镜则是用两个光子来激发同一个荧光分子.与单光子显微镜相比,它所使用的激光波长更长(单个光子的能量更小),具备更高的组织穿透性和更小的光毒性.此外,双光子的激发能力与能     

可用于双色双光子显微成像的新的荧光蛋白对

双色双光子激光扫描显微技术可以用来研究生物组织内两种不同蛋白质的表达、定位和示踪．由于大多数双光子显微镜一次只能提供一种波长的激发光,双色同时成像较难实现．mAmetrine和mKate2作为新发现的荧光蛋白对可以用于双光子双色同时成像,这得益于它们各自的优势：mAmetrine的斯托克斯位移和mKate2的高亮度．在765nm的波长激发时,它们的双光子吸收效率都很高．mAmetrine和mKate2能够很好地用于双色双光子活细胞成像实验．     

双光子显微成像系统群延迟色散的测量和补偿

为了对双光子显微成像系统的群延迟色散进行校正,提高双光子激发效率的目的,采用自相关仪测量的方法在自行搭建的双光子系统光路的四个位置测量飞秒激光的脉冲展宽情况,测量样品位置5个波长下最优的群延迟色散补偿值,由此拟合得到自搭建双光子系统的全波段群延迟色散补偿曲线。实验结果表明在应用此群延迟色散补偿曲线后样品位置的脉冲宽度平均减小95 fs,在两个典型激发波长(750 nm和900 nm)生物样品的荧光强度分别提高了42.7%和76.8%。结论为双光子激发效率与飞秒激光的脉冲宽度成线性反比关系。     

活体细胞内双光子激发的光漂白特性

长波长光的强穿透能力和对活体细胞和生物组织光毒性很小的特性,使得双光子激发荧光显微术已经成为无损伤成像的重要工具.可是双光子激发的高光子密度可能会产生高次光子相互作用, 从而产生更快的光漂白.从实验上研究了离体和活体细胞内的若丹明123和若丹明B分别在单光子激发和双光子激发时的光漂白特性.在体的实验结果与离体的实验结果一致.正如期望的一样,单光子激发时光漂白速率非常近似地随着激发功率的增加而线性增加.可是,双光子激发时的光漂白速率并不是正比于激发功率的平方,而是正比于激发功率的高次方（＞3.5）.对绿色荧光蛋白（GFP）变异体CFP和YFP的实验也得到同样的结果,这就表明高次光漂白可能是双（多）光子激发中的普遍现象.因此多光子的应用可能会受到强光漂白的限制.     

光生物学与光医学中的新技术及其应用

近几年内,光子生物学与光子医学发展非常快,本文主要从四个方面介绍了近期内在光子生物学与光子医学领城内取得的重要进展：(1)双光子技术,可检测胚胎活组织、确定生物的非损伤激发光阈值、对人体肌纤维进行三维成像;(2)光镊技术,用于研究细胞的应变能力、细胞膜的弹性、跟踪并描述单个分子之间的结合以及操纵DNA分子;(3)光学探针技术,检测疾病、研究构象变化;(4)光学成像技术,主要集中介绍对肌动蛋白的成像方面。     

生物分子的双光子激发发光

应用Nd:YAG激光器和光学光谱分析仪对蛋白质分子的同时吸收双光子过程作了进一步研究,讨论了蛋白质分子双光子过程的特性和估计了它们的双光子吸收截面,并得到胰蛋白酶,白蛋白和色氨酸等分子由于双光子激发产生的荧光光谱.     

生物光子学应用

生物光子学是光学技术与生命科学的交叉学科,可以在分子水平上研究细胞的功能和结构,包括生物系统的光子辐射以及这些光子携带的信息,用光子及其技术对生物系统进行检测、加工和改造等。激光扫描共焦显微术、双光子荧光显微术、近场光学扫描显微术和光镊等显微技术在生命科学研究中的应用非常重要。     

深层组织双光子荧光成像激发特征的仿真分析

双光子荧光探针被广泛研究应用于生物医学成像和治疗,深入了解这类探针在生物组织中激发分布特征及其相对于单光子探针的成像优势和劣势对探针的合理选择和应用具有指导意义。然而,由于实际测量难以避开众多干扰因素的影响,因此不能准确反映其固有特征。本文选取典型生物和荧光激发参数,利用生物仿真定量分析,并通过双光子和单光子的激发成像对比,获取了双光子荧光的基本激发特征。结果发现,在相同平圆光束激发下,双光子激发由于激发效率低且需要双光子吸收,再加上较高的激发阈值,虽然激发光在组织穿透深度方面存在优势,但在组织内衰减速率快、有效穿透深度比高量子效率的单光子荧光激发浅。同时,深度方向的快速衰减会导致横向激发光能的非均匀性分布,这种非均匀性对双光子荧光影响更大。仿真分析进一步表明:在生物组织耐受的前提下提高双光子激发的功率更有利于荧光成像;增加光照半径可以减弱横向激发的不均匀性,从而改善双光子荧光成像的效果。本研究结果为双光子荧光激发、成像的应用提供了基础数据参考,本仿真方法也为快速研究光与生物组织的相互作用提供了借鉴。     

双光子激发荧光各向异性度的成像

荧光各向异性度 (fluorescence anisotropy) 测量可以获得荧光分子的转动速度信息,进而了解分子质量、结构、以及与周边环境的相互作用情况 . 围绕一台双光子激发扫描荧光成像系统,通过改变外光路和图像记录与处理程序,从而实现了双光子激发荧光各向异性度成像,并针对一些典型样品和体系,展示了该方法的应用 . 实验中观察了 FITC 荧光分子、 FITC 结合的 CD44 抗体分子及与肿瘤细胞表面受体结合的 FITC-CD44 抗体分子 . 测量结果表明,不同分子质量、不同微观环境状态下的荧光分子,其各向异性度大小不同,在各向异性度图中能够被明显区分 . 荧光各向异性度成像能够定量测量样品微区的各向异性度值,并以二维图像的形式直观表达,是各向异性度测量与成像技术的良好结合 .     

离轴抛物镜扫描中继系统提升双光子显微成像视场

本文提出利用离轴抛物镜共焦中继系统来改善双光子成像视场边缘像质变差的问题,进而提升双光子成像视场。不同于传统的双胶合透镜扫描中继系统,离轴抛物镜共焦扫描中继系统由一对离轴抛物镜构成,扫描振镜位于抛物面镜焦点处。仿真和试验结果均显示,该系统能有效优化视场边缘的像散、场曲和畸变情况,提高成像质量。利用该扫描系统,我们实现了视场为2. 4 mm×2. 4 mm,横向分辨率为1μm的大视场双光子显微成像,能清晰分辨小鼠大脑切片中的神经轴突结构。     

Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy

We have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equipping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double-labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single-molecule level.     

The effect of fluorescent labeling on calcium-induced fusion of fusogenic carrot protoplasts

Various fluorescent compounds — carboxyfluorescein, scopoletin, fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), rhodamine 123, and rhodamine B ethyl ester — were used to study their effects on calcium-induced fusion of fusogenic carrot (Daucus carota L.) protoplasts. These protoplasts normally fused at a high percentage (50–60%) in response to 10 mM calcium, pH 6.0; however, if cells had been labeled with scopoletin, FITC, or RITC, fusion was greatly reduced. In contrast, labeling with carboxyfluorescein, rhodamine 123, or rhodamine B ethyl ester had no detectable effect on calcium-induced fusion. The two rhodamine dyes are shown to be localized in mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - FITC fluorescein isothiocyanate - RITC rhodamine isothiocyanate - PE phosphatidylethanolamine     

Sulfadimethoxine and rhodamine B as oral biomarkers for European badgers (Meles meles)

A field study was carried out on Little Island (County Waterford, Ireland) in June 2000 to evaluate the potential of a bait-marking system for use in European badgers (Meles meles). Two oral biomarkers, sulfadimethoxine (SDM) and rhodamine B, were incorporated into fishmeal baits and distributed by hand at main sets in five test territories for 3 consecutive days. In parallel, non-biomarked baits were distributed at a single control territory. The objectives of the study were to: (1) assess the effects of SDM and rhodamine B on palatability and thus bait acceptance, and (2) investigate the marking capacity of SDM and rhodamine B in serum and hair samples taken from badgers. Trapping was carried out in each territory for 5 consecutive days immediately after bait distribution. Analysis of data revealed that 90-100% of baits were removed in four of the test territories and from the control territory. In the fifth test territory, 61% of baits were removed. Of the badgers (n = 26) trapped in the test territories, 18 (69%) were positive when tested for both biomarkers. In contrast, the remaining eight animals and those captured in the control territory (n = 6 badgers) were negative. In the marked animals, the highest levels of SDM were recorded in serum samples taken soon after bait distribution. Thereafter, the levels declined in each badger over the course of the study. In contrast, rhodamine B was readily detectable by fluorescence microscopy of hair samples throughout the period of study. The results indicate that SDM and rhodamine B act as systemic markers in badgers and have potential future applications for monitoring of oral vaccine uptake.     

Self-association of glucagon as measured by the optical properties of rhodamine 6G

The fluorescence of rhodamine 6G is completely quenched in glucagon solutions in 0.6 M K2HOP4 at pH 10.6. The absorption of rhodamine 6G is red-shifted by the same reaction. A single rhodamine 6G molecule appears to be bound to a hydrophobic patch in the center of the trimer of glucagon. Since the glucagon monomer has almost no organized structure this site exists only in the associated trimer form of glucagon. The self-association of glucagon to the trimer has been determined from the variation in rhodamine 6G fluorescence and absorption measured over a 60-fold range of dye concentration. The self-association constant agrees with values determined by other methods in the absence of dye. The binding isotherms of rhodamine 6G to glucagon shift with glucagon concentration and exhibit negative cooperativity.     

Inorganic phosphate determination: Colorimetric assay based on the formation of a rhodamine B-phosphomolybdate complex

A sensitive technique for inorganic phosphate determination was developed. It is based on the formation of an insoluble rhodamine B-phosphomolybdate complex. After it is washed with 1 HCl the precipitate is dissolved in acetone and rhodamine B is measured spectrophotometrically at 555 nm. In 1 HCl, the complex is composed of three molecules rhodamine B and one molecule phosphomolybdate. Due to the high molar absorbance of rhodamine B in acetone and to the threefold amplification of dye concentration compared to P_i concentration in the precipitated complex, a molar absorption coefficient of 330,000 ± 5000 ⁻¹ cm⁻¹ (SD) is obtained. This allows the determination of quantities as low as 1.5 nmol P_i with good precision, while quantities as low as 0.5 nmol P_i are detectable. The effect of anions and buffers was studied. Some possible applications of the method are illustrated, as, e.g., enzyme activity measurement at very low substrate concentration and determination of small quantities of P_i and total phosphate in (biological) samples.     

Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells

In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was established. Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential. P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell-rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1. The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria. The kinetics of decrease in rhodamine B fluorescence (V(i)) can be used to estimate DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46. The DeltaPsi(m) values determined were -160+/-4 mV for K562 cell, -146+/-6 mV for K562/adr cell, -161+/-10 mV for GLC4 cell and -168+/-2 mV for GLC4/adr cell. An increase or a decrease in DeltaPsi(m) consequently followed an increase or a decrease in the cellular ATP contents. An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.     

Structural relationship between "glutamic acid" and "lysine" forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.

Urokinase digestion of maleinated plasminogen results in cleavage of the single peptide bond Arg-68-Met-69, which is one of the bonds normally cleaved during the first step of the activation procedure. The inactive intermediate compound formed in this way was subjected to NH2-terminal amino acid sequence analysis, which clearly demonstrates the structural relationship between the forms of plasminogen with different NH2-terminal amino acids. It is thus shown that lysine-78 and valine-79 in the "glutamic acid" plasminogen actually are the NH2-terminal amino acids in "lysine" and "valine" plasminogen respectively. The forms with glutamic acid in NH2-terminal position are called plasminogen A, while all other forms lacking the NH2-terminal part of the molecule and which can be activated in a single step are called plasminogen B. By affinity chromatographic studies of the NH2-terminal activation peptide on insolubilized plasminogen B, it was demonstrated that this peptide has specific affinity for plasminogen B. It was also shown that this noncovalent interaction is broken by 6-aminohexanoic acid in two concentration. The tryptic heptapeptide (Ala-Phe-Gln-Tyr-His-Ser-Lys) which occupies the positions number 45 to 51 in the NH2-terminal activation peptide (as well as in the intact plasminogen molecule) is importance for the conformational state of the plasminogen molecule.     

Two-photon thermal bleaching of single fluorescent molecules

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.     

Fluorescence correlation spectroscopy as a tool to investigate single molecule probe dynamics in thin polymer films

Fluorescence correlation experiments were performed on rhodamine 6G in PDMS spin-coated films on glass surfaces. With polarised excitation, ensemble bleaching of the dye and single molecule intensity fluctuations were observed. From the statistics of single molecule intensity data taken at different positions in the film, correlation functions were calculated. Two modes of motion with exponential decay shapes and correlation times of tau(c) = 0.15 s and tau(c) = 0.7 s could be detected. Potential origins of intensity fluctuations are lateral diffusion, rotational diffusion or intramolecular fluctuations of dyes involving spectral diffusion or photoinduced processes. From the experimental results, lateral diffusion can be ruled out as a motional mode. Single molecule fluctuations are assigned to changes of the molecular configuration of the dyes, which are rigidly bound to the glass. To assess the environmental influence on such molecular motions, the bulk viscosity of the PDMS was varied over two orders of magnitude, leading to changes of tau(c) of the slow mode by a factor of four. This result proves the sensitivity of the single molecule fluctuations to the molecular scale dynamics of the surrounding polymer matrix and makes the correlation time a measure of the local environment of dye probes.     

Ratiometric fluorescent detection of Cu2+ based on dual‐emission ZIF‐8@rhodamine‐B nanocomposites

Zeolitic imidazolate framework‐8 (ZIF‐8) loading rhodamine‐B (ZIF‐8@rhodamine‐B) nanocomposites was proposed and used as ratiometric fluorescent sensor to detect copper(II) ion (Cu²⁺). Scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray powder diffraction, nitrogen adsorption/desorption isotherms and fluorescence emission spectroscopy were employed to characterize the ZIF‐8@rhodamine‐B nanocomposites. The results showed the rhodamine‐B was successfully assembled on ZIF‐8 based on the π‐π interaction and the hydrogen bond between the nitrogen atom of ZIF‐8 and –COOH of rhodamine‐B. The as‐obtained ZIF‐8@rhodamine‐B nanocomposites were octahedron with size about 150–200 nm, had good water dispersion, and exhibited the characteristic fluorescence emission of ZIF‐8 at 335 nm and rhodamine‐B at 575 nm. The Cu²⁺ could quench fluorescence of ZIF‐8 rather than rhodamine‐B. The ZIF‐8 not only acted as the template to assemble rhodamine‐B, but also was employed as the signal fluorescence together with the fluorescence of rhodamine‐B as the reference to construct a novel ratiometric fluorescent sensor to detect Cu²⁺. The resulted ZIF‐8@rhodamine‐B nanocomposite fluorescence probe showed good linear range (68.4 nM to 125 μM) with a low detection limit (22.8 nM) for Cu²⁺ combined with good sensitivity and selectivity. The work also provides a better way to design ratiometric fluorescent sensors from ZIF‐8 and other fluorescent molecules.