多焦点多光子显微技术及其研究进展

多焦点多光子显微技术(multifocal multiphoton microscopy,MMM)提高了激发光能的利用率和成像速度,可以实现样品的三维快速多光子激发荧光显微成像,并具有对活体样品损伤小,成像深度大,图像信噪比高等优点.详细阐述了MMM的实现方法及其研究进展,包括同时时间和光谱分辨的MMM(simulta...     

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.     

基于双光子激发的荧光相关谱测量

本文利用多光子激发激光扫描显微镜的部分光路和探测器．建立了双光子荧光相关谱系统(Two-Photo Fluorescence Correlation Spectroscopy．简称TP-FCS)。利用TP-FCS系统观察到了“光子爆发”现象．实现了染料分子的双光子激发,测量出若丹明B染料分子在蔗糖溶液中的扩散系数。实验证明该系统具有操作简便、可靠性高,费用低廉等等点,可实现单分子检测。     

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     

生物科学研究中的激光多光子显微技术

本文综述了生物科学中近红外（NIR）多光子激光扫描显微技术的进展,其中包括：多光子显微特点,激光光源,荧光寿命的测量,多光子多色实时杂化荧光（FISH）,非人侵性活体光学解剖,植物学中的多光子显微技术,细胞的多光子显微损伤、皮米非入侵和非接触性外科手术等。     

多光子显微镜在研究阿尔茨海默病中的应用

多光子显微镜采用近红外线激发荧光成像,能直接观察AD老年斑的自然病理进展,并且在鼠模型中进行抗老年斑治疗的疗效评估.其高分辨率的特点,可用于监测蛋白与蛋白之间、蛋白构象改变和蛋白水解而产生的荧光共振能量转移(FRET)改变.     

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。     

上转换发光成像技术与靶向成像应用

上转换发光是指稀土离子吸收两个或两个以上低能光子(近红外光)而辐射一个高能光子(可见光)的发光现象。与传统紫外激发相比,上转换发光由于采用近红外光激发而具有高的组织穿透深度、弱的生物样品损伤且无生物样品自发荧光,这些优点表明上转换发光在生物成像方面具有广阔的应用前景。文章介绍了基于稀土上转换发光过程的显微成像技术和活体成像技术,及其在肿瘤靶向成像领域的应用。     

鸡胚完整脑的光子发射和光激发光研究

利用自制的单光子记录系统,对离体的浸于DMEM培养液中的鸡胚的完整脑所进行的光子发射测量显示出整脑与培养液相比有光子发射.完整脑的光子发射强度与胚龄,鸡胚的健康状况和离体后脑组织的新鲜程度有关.在对完整脑的光激发光测量中观察到一种短寿命的延迟发光,它的衰减过程不服从指数衰减规律而表现出双曲线衰减行为.从生物光子发射归因于生物体内的非定位的相干性电磁场及Frohlish的生命系统中存在着长距离的相干性的理论,讨论了利用生物光子发射研究细胞间通讯、生物的调节及生物与环境关系的意义,以及所检测的光子发射与生物系统自身的相干性电磁场的关系,并提出应从环境对生物系统自身场的影响来理解所测的生物光子发射.     

多光子显微成像技术在脑部疾病研究中的应用

由于多光子显微技术具有高时空分辨率、低损伤性、可对活体长时间成像等特点,近年来已被广泛应用于生物医学等领域,并且在多种疾病诊断中展现出巨大的应用潜力.尤其是在脑部疾病的研究中,利用多光子成像技术可实现对复杂神经网络的研究,包括对脑部神经细胞、血管、肿瘤等进行实时成像并研究各自之间的相互作用,能进一步揭示脑疾病的发病机制并指导检测治疗方法的开发.本文简要介绍了多光子成像技术的基本原理及特点,总结了其在阿尔茨海默病、脑中风、脑肿瘤等多种脑部疾病中的应用,详细阐述了近年来利用多光子成像技术在脑部疾病研究中所获得的成果,并对多光子成像技术的发展前景进行了展望,预期其在脑部疾病的研究中将发挥更大的作用.     

Improved identification of methanogenic bacteria by fluorescence microscopy.

Methanogenic bacteria can be tentatively identified by fluorescence microscopy. This technique was improved by carefully selecting a series of excitation and barrier filters that matched the excitation and emission spectra of some unique coenzymes viz., F420 and F350, in methanogenic bacteria.     

Improved identification of methanogenic bacteria by fluorescence microscopy.

Methanogenic bacteria can be tentatively identified by fluorescence microscopy. This technique was improved by carefully selecting a series of excitation and barrier filters that matched the excitation and emission spectra of some unique coenzymes viz., F420 and F350, in methanogenic bacteria.     

Electric field allowed molecular transitions for one and two photon excitation microscopy

We propose an excitation technique for observing single and two photon excitation in those molecules for which such transitions are forbidden by the selection rules. This is possible by the application of an external electric field that perturbs the molecular orbitals, thereby resulting in a significant shift of energy levels. Such a shift of energy levels may bring those levels in resonance with the radiation field which is normally forbidden by selection rules. Further, parity of the these states may significantly improve the emission process. The external electric field results in the mixing of excited (short lifetime) and metastable states (long lifetime), thus reducing the lifetime of metastable (or near metastable) states. This may provide an effective channel for allowing transition from the metastable states. An application of electric field may result in the excitation of poorly excitable biomolecules. This excitation technique may find applications in single- and multi-photon fluorescence microscopy, bioimaging and optical devices.     

X-ray fluorescence analysis using synchrotron radiation at the photon factory

X-ray fluorescence experiments at the Photon Factory in Japan are described. An energy-dispersive X-ray fluorescence system was combined with various excitation modes, i.e., a continuum and a monochromatic excitation, which consist of a crystal monochromator or a wide band pass monochromator. These excitation modes provide a wide range of band width and photon flux of excitation beams. Minimum detection limits obtained for a thin sample were less than 0.1 ppm and 0.1 pg when there was no line interference. Advantages of using monochromatic excitation are discussed, with emphasis on the possibility of chemical state analysis. Grazing incidence X-ray fluorescence is a technique very appropriate to synchrotron radiation characteristics. Near-surface analysis and trace analysis of solution samples placed on a total reflection support were made. Future plans are discussed, including microbeam analysis, tomography, X-ray excited optical fluorescence, and applications of insertion devices (undulator and wiggler).     

Two-photon excitation in fluorescence polarization receptor-ligand binding assay

Fluorescence polarization is one of the most commonly used homogeneous assay principles in drug discovery for screening of potential lead compounds. In this article, the fluorescence polarization technique is combined with 2-photon excitation of fluorescence. Theoretically, the use of 2-photon excitation of fluorescence increases the volumetric sensitivity and polarization contrast of fluorescence polarization assays. The work in this report demonstrates these predictions for an estrogen receptor ligand binding assay.     

Is Metaphit a phencyclidine antagonist? Studies with ketamine, phencyclidine and N-methylaspartate

The dissociative anaesthetics, phencyclidine and ketamine, block excitation of central neurones by N-methylaspartate. Using the technique of microelectrophoresis on rat spinal neurones in vivo Metaphit, a phencyclidine receptor acylating agent, was tested to see whether it would antagonise this effect of dissociative anaesthetics. The predominant effect of Metaphit was, however, to reduce N-methylaspartate induced excitation. It is concluded that Metaphit has mixed agonist/antagonist effects at the phencyclidine receptor.     

植物光诱导延迟荧光测量的影响因素研究

光合作用是地球上最重要的化学反应,植物内源性光诱导延迟荧光是光合作用原初过程中光系统Ⅱ作用中心P680处电荷分离效率的内在探针。延迟荧光除了受植物本身及其生长发育状况有关外,还受到其他很多环境及测量方面的影响,所以为了更好地利用延迟荧光特性技术研究植物生理特性,就必须对测量参数指标做合理的优化。本文从影响延迟荧光的激发光源的光强,激发时间及外界环境温度出发,研究延迟荧光的变化特性,为延迟荧光在植物生理特性方面的研究提供合理的理论依据。     

Two-color immunofluorescence using a fluorescence-activated cell sorter.

A technique for the analysis of fluorescein and rhodamine in a flow system using a single wavelength of excitation is described. Both optics and electronics are used to discriminate the fluorescein and rhodamine signals. This technique has been used to study the relationship between immunoglobulin M and immunoglobulin D on mouse splenic lymphocytes.     

Cross talk free fluorescence cross correlation spectroscopy in live cells

Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin.     

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.