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。     

Photobleaching with a subnanosecond laser flash

In standard fluorescence recovery after photobleaching (FRAP) applications for measuring lateral diffusion rates and adsorption/desorption kinetics of fluorescent molecules at biological or model membranes, irreversible bleaching is induced by a bright excitation flash of at least millisecond time scale. It has been presumed that the bleaching event is of a low probability and the significant bleached population that develops during the flash results from each molecule undergoing thousands of excitation/deexcitation cycles before a bleaching event occurs. In some FRAP experiments, notably polarized FRAP (PFRAP) for measuring molecular rotational diffusion rates, it is desirable to use much shorter (subnanosecond) bleaching pulses. However, subnanosecond pulses are shorter than the fluorescence lifetime, so that any fluorophore will experience at most only one visit to the excited state during the bleaching pulse. If bleaching occurs only by the same processes as in slower FRAP experiments, one would thereby expect only minimal bleaching regardless of the bleach intensity. Moreover, the ability of fast polarized pulses to imprint an anisotropic orientational pattern in the postbleach unbleached fluorophore, an ability essential for PFRAP, is not at all guaranteed, particularly if two-photon processes are involved in high-intensity short bleach pulses. In this study, bleaching depths are measured as a function of subnanosecond pulse intensity on a small labeled protein covalently immobilized on fused silica. We show that bright subnanosecond laser flashes do indeed produce significant bleaching, that both two photon effects and reversible bleaching are involved, and that polarized bleaching does produce an anisotropic orientational pattern of unbleached fluorophore. We also postulate a theoretical molecular state model which semiquantitatively accounts for the experimentally observed dependence of reversible bleaching on bleaching pulse intensity.     

Improved Fluorescent Proteins for Single-Molecule Research in Molecular Tracking and Co-Localization

Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and > 50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores. Authors contributed equally to this article.     

Antifading embedding media in confocal immunoflourescence microscopy

Fading or bleaching of fluorescence intensity during continuous illumination of stained objects is a serious problem in fluorescence microscopy. Fluorescence intensity as well as bleaching characteristics of dyes are dependent primarily upon physical parameters such as molecular constants (absorption rate and quantum efficiency), excitation energy and brightness (causes photon saturation), and environmental parameters (pH, ions, binding to proteins, etc.) that can strongly influence the properties of fluorochrome molecules. We have studied the effect of various antifading reagents on the behavior of the common dyes fluorescein isothiocyanate (FITC) and phycoerythrin (PE) using immunofluorescent-stained living cells in suspension or membrane-permeabilized dried cells as test systems. As expected, fading cannot be completely eliminated but may be reduced to varying degrees. In our hands, the most efficient antifading reagent for FITC isn-propyl gallate (NPG) dissolved in glycerol. No additive was found to retard fading, but complete dehydration of the cell suspension reduces this effect.     

Three-photon induced photobleaching in a three-dimensional memory material

The photobleaching properties of a 3D memory material based on pyrylium salts are studied. It is shown that under ultrashort pulsed irradiation photobleaching occurs through two diverse mechanisms. One is major and causes fast bleaching, whereas the other is minor and causes slow bleaching. Furthermore, it is proved that both mechanisms are three- or more-photon processes. This fact gives the capability of significantly increasing data storage density.     

Photobleaching and stabilization of. fluorophores used for single-molecule analysis. with one- and two-photon excitation

Fluorescence spectroscopic measurements at the single-molecule level usually require large absorption cross sections and fluorescence quantum yields for the dyes under study. In addition to these parameters, the collectable number of fluorescence photons and, thus, the signal-to-noise ratio of the measurement, is influenced by processes like triplet-state population and photobleaching, shifting the saturation threshold of the dye to lower excitation intensities. Confocal fluorescence correlation spectroscopy (FCS) is a versatile method to precisely determine photon emission rates of single molecules but also gives access to rate constants of dynamic bleaching and intersystem crossing. In recent FCS studies in solution and living cells, two-photon excitation with its inherent spatial sectioning has proven to be a very valuable alternative to minimize background and cumulative signal loss. However, there is evidence that in many dye systems, the photobleaching rates with two-photon excitation are significantly enhanced with respect to one-photon excitation at comparable photon-emission yields. The reasons have so far remained mainly speculative. In the present study, potential photobleaching pathways are investigated by adding chemical stabilizers and by working at different oxygen concentrations. The results suggest that the population of triplet states does not appear to be responsible for the limited emission rate with two-photon excitation. Rather, photobleaching pathways via the formation of radicals seem to be plausible causes for the signal limitation. Favorable conditions are discussed to maximize the overall photon-collection yield in two-photon experiments. Received: 4 July 2001 / Published online: 23 November 2001     

Superconducting characteristics of (Bi, Pb)-2223 single crystals with controlled oxygen-content

Critical current density was measured for oxygen-controlled (Bi, Pb)-2223 single crystals before and after the irradiation with gold ions in a magnetic field parallel to the irradiation-induced defects along the c-axis. Eleven specimens prepared in different annealing conditions were measured. The condensation energy density of each specimen was evaluated from the observed critical current density by using the summation theory of pinning forces of columnar defects and the flux creep theory. It was found that the specimen heat-treated at 1 atm in oxygen atmosphere has the highest condensation energy density among all specimens. Hence, it is speculated that the optimum oxygen pressure for the anneal is around 1 atm.     

双光子技术的应用研究进展

双光子技术在未来光电子集成、生物分子探测、医学诊断等领域具有巨大应用潜力和广阔应用前景。文章着重介绍了双光子过程在荧光显微成像、高密度数据存储以及微细加工等领域的应用研究进展。对影响双光子技术的应用和发展的相关领域的研究动态也进行了简要讨论。     

A simple x‐ray monochromator based on an alligator lens

Alligator lenses, i.e. two inclined arrays of sawteeth or prisms, which face each other, can focus x‐rays with photon energies >4 keV. The inclination angle can be changed easily, and thus either the focal length for fixed photon energy or the photon energy in a fixed slit position can be varied. The material distribution in this condition is approximately parabolic along the teeth axis, hence it is of the shape required for aberration‐free focusing at a given photon energy. As the refractive index varies significantly with photon energy in the x‐ray range, these lenses suffer from chromatic aberrations, if illuminated with white x‐rays. In combination with a slit such a lens can therefore be used as an easily insertable inline monochromator. In this work, a simple universal function for the dependence of the transmission on the photon energy was derived for this application. The required tolerances for the shape of the sawteeth are found to be compatible with standard workshop machining procedures. A laboratory‐made lens of Plexiglas is shown to increase the flux density in a laboratory setup by a factor of 3, i.e. 50% of the expected result for a perfect lens. The discrepancy can be consistently ascribed to macroscopic defects of the sawteeth tips. Expectations for the performance of these lenses as monochromators at synchrotron radiation sources are presented. A single Be alligator lens is expected to provide tuning between at least 8 keV and 20 keV photon energy with a bandpass of 6%, sufficient for XRF and SAXS experiments. Consequently, such a lens pair is all that is needed for building simple synchrotron radiation beamlines for special x‐ray experiments. Copyright © 2004 John Wiley & Sons, Ltd.     

Photostability comparison of CdTe and CdSe/CdS/ZnS quantum dots in living cells under single and two-photon excitations

The photostability is an outstanding feature of quantum dots (QDs) used as fluorescence probes in biological staining and cell imaging. To find out the related factors in the QD photostability, the photobleaching of naked CdTe QDs and BSA coated CdSe/CdS/ZnS QDs in human hepatocellular carcinoma (QGY) cells and human nasopharynx carcinoma (KB) cells were studied under single photon excitation (SPE) and two-photon excitation (TPE). In these two cell lines the cellular QDs were irradiated by a 405 nm continuous wave laser for SPE or an 800 nm femto-second (fs) laser for TPE. The QD photobleaching with the irradiation time was found to fit a biexponential decay. The fast decay plays a dominant role in the bleaching course and thus can be used as the parameter to quantitatively evaluate the QD photostability. The TPE decreased the QD photobleaching as compared to SPE. The BSA coated core/shell QDs had improved the photostability up to 4-5 times than the naked QDs due to the shielding effect of the QD shell. Therefore, it is better to use core/shell structured QDs as the fluorescence probe combining with a TPE manner for those long-term monitoring studies.     

X射线探测器的脉冲标定技术

利用强流电子束技术产生通量密度为10¹⁸—10¹⁹X-ray photon/sr·s的脉冲CuKX射线源,标定PIN型硅二极管半导体探测器对X光子的脉冲灵敏度。用绝对X射线监测器——P10气体脉冲电离室作为脉冲X射线通量密度的标准。脉冲电荷自动测量仪由微处理机进行程序控制,并予以实时校准。该电离室测量通量密度的精度为±5％,适用的能通量率范围可达4×10^-9—2×10²W/cm²,适用的光子能量范围为1.5—10keV,标定探测器的精度为±7.0％,并发现PIN型硅二极管的脉冲灵敏度比稳态X射线束标定的灵敏度高30％左右。 关键词：     

Diode-pumped doubly Q-switched Nd:LuVO4 laser with AO modulator and GaAs saturable absorber

A diode-pumped doubly Q-switched Nd:LuVO₄ laser with an acousto-optic (AO) modulator and GaAs saturable absorber has been realized. The pulse profile of the doubly Q-switched Nd:LuVO₄ laser has an almost absolutely symmetric shape. The dependences of pulse width, single-pulse energy and peak power on the incident pump power under different AO repetition rates are measured. By considering the transverse Gaussian distribution of the intracavity photon density and the longitudinal distribution of photon density along the cavity axis, the coupled rate equations of the laser are given. These coupled rate equations are solved numerically and the theoretical results are in agreement with the experimental results.     

Gamma or X-rays attenuation properties of some biochemical compounds

The probability of gamma or X-ray interactions with important 14 antioxidants have been discussed for total photon interactions in the wide energy range of 1?keV–100?GeV using the WinXCOM code. The variations of mass attenuation coefficient (μ_ρ), effective atomic number (Z_eff) and electron density (N_el) with photon energy were plotted for total photon interactions. It was found that the values of μ_ρ, Z_eff and N_el depend on the incoming photon energy and chemical compositions of antioxidant. The highest values of these parameters were found at a low-energy zone where the photoelectric effect is the dominant interaction process. When antioxidants were compared with each other, it was seen that Z_eff has the highest values for Oenin chloride and Delphinidin chloride which contain the Cl element. This investigation is thought to be useful for medical applications where radiation exposure is present.     

基于DVD光头的双光子光致漂白三维光存储

基于现有的DVD光头物镜与音圈电机，根据光致漂白的双光子吸收气维光信息存储原理，以钛蓝宝石飞秒脉冲激光进行双光子光信息写入和读出，利用音圈电机进行选层，在新型光致漂白材料二苯乙烯衍生物中进行光致漂白二进制编码信息的存储和读出实验研究；实现了三层光信息存储，信息点间距和信息层间距分别为4μm和15μm；用Matlab软件读出信息的信号强度并对其进行了识别，识别结果与写入的二进制编码信息完全一致。实验证明了用DVD光头进行双光子三维光数据存储的可行性，表明双光子吸收光致漂白技术可以与现有CD／DVD兼容，为实现多层高密度和超高密度光信息存储打下基础。     

X光辐射在泡沫介质中超声速传输研究进展

X光辐射输运过程通过光子的发射与吸收使能量在介质内进行再分配,而光子的辐射和吸收过程对从介质内出射的能谱产生十分显著的影响。辐射输运由积分-微分方程来描述,得到它的解极其困难,这是因为此方程依赖于局域和非局域的条件。当外加的非局域的X光辐射场作用在介质上一个强的X光辐射能流时,介质的局域温度和密度将影响X光的吸收和发射。本文将对辐射输运的方程和基本理论进行阐述,并对辐射超声速传输的实验结果进行评述。首先,介绍辐射在介质中的传输的理论基础以及简化分析模型;其次,对辐射在介质中扩散超声速输运进行解析分析,我们首次导出辐射的超声速传输条件下的辐射能流与物质能流之比与马赫数和光学厚度的定量关系;最后,介绍国外的主要实验结果,同时也给出我们近期的研究结果。我们的实验结果表明,不同能区的光子因辐射不透明度不同使得在介质中的传播时间不同,并且实验测出光学厚度。     

X光辐射在泡沫介质中超声速传输研究进展

X光辐射输运过程通过光子的发射与吸收使能量在介质内进行再分配,而光子的辐射和吸收过程对从介质内出射的能谱产生十分显著的影响。辐射输运由积分-微分方程来描述,得到它的解极其困难,这是因为此方程依赖于局域和非局域的条件。当外加的非局域的X光辐射场作用在介质上一个强的X光辐射能流时,介质的局域温度和密度将影响X光的吸收和发射。本文将对辐射输运的方程和基本理论进行阐述,并对辐射超声速传输的实验结果进行评述。首先,介绍辐射在介质中的传输的理论基础以及简化分析模型;其次,对辐射在介质中扩散超声速输运进行解析分析,我们首次导出辐射的超声速传输条件下的辐射能流与物质能流之比与马赫数和光学厚度的定量关系;最后,介绍国外的主要实验结果,同时也给出我们近期的研究结果。我们的实验结果表明,不同能区的光子因辐射不透明度不同使得在介质中的传播时间不同,并且实验测出光学厚度。     

Radiation damage in soft X-ray microscopy

The rates of chemical transformation by radiation damage of polystyrene (PS), poly(methyl methacrylate) (PMMA), and fibrinogen (Fg) in a X-ray photoemission electron microscope (X-PEEM) and in a scanning transmission X-ray microscope (STXM) have been measured quantitatively using synchrotron radiation. As part of the method of dose evaluation in X-PEEM, the characteristic (1/e) sampling depth of X-PEEM for polystyrene in the C 1s region was measured to be 4 ± 1 nm. Critical doses for chemical change as monitored by changes in the X-ray absorption spectra are 80 (12), 280 (40) and 1230 (180) MGy (1 MGy = 6.242*ρ eV/nm³, where ρ is the polymer density in g/cm³) at 300 eV photon energy for PMMA, Fg and PS, respectively. The critical dose for each material is comparable in X-PEEM and STXM and the values cited are thus the mean of the values determined by X-PEEM and STXM. C 1s, N 1s and O 1s spectroscopy of the damaged materials is used to gain insight into the chemical changes that soft X-rays induce in these materials.     

Fluorescence Recovery Under Decaying Photobleaching Irradiation: Concept and Experiment

A novel modification of photobleaching method for measurement of lateral diffusion is developed. In this approach fluorescence recovery kinetics is measured under decaying photobleaching irradiation, termed as fluorescence recovery under decaying photobleaching (FRDP). The time evolution of fluorescence intensity normalized to input irradiation starts from the photobleaching kinetics and transforms into the kinetics of fluorescence recovery at a later stage resulting in appearance of minimum. The analytical solution for the kinetics of fluorescence for Gaussian lineshape of laser beam and hyperbolic decay of irradiation in the first order approximation on bleaching rate was obtained. The accuracy of the analytical function was evaluated with exact numerical solution computed with finite differentiates method. The FRDP method was successfully applied to fluorescein solution in the glycerol/water mixture (80%) under various experimental settings using home-made experimental set-up. The FRDP approach demonstrated 25–30 fold enhancement in signal intensity over classical fluorescence recovery after photobleaching (FRAP) method at 3–5 fold increase in total irradiation. Among other advantages of the FRDP is the opportunity to perform measurements on varying time scales under constant size of the bleaching spot, including “safe” long time measurements. The potential extra advantage of FRDP method for analysis of complex diffusion in the biological system is discussed.     

Single-Photon Counting Multicolor Multiphoton Fluorescence Microscope

We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV–Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10⁸ counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte -stacks composed of 16 spectral channels, 256× 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.     

Resonant Photon Drag of Dipolar Excitons

The theory of the photon drag of dipolar excitons in double-quantum-well nanostructures is presented. It is shown that the exciton-drag flux density features a resonant behavior if the photon frequency is close to some transition frequency in the discrete exciton spectrum. When the structure is irradiated with polarized light, the resonant enhancement of the drag current occurs when the photon energy coincides with the energy of an excited level of the exciton internal motion and the components of the angular momentum of internal motion in the initial and final states differ by one. The proposed effect can be used to control exciton transport in nanostructures based on a two-dimensional exciton gas.