Alterations of single molecule fluorescence lifetimes in near-field optical microscopy

Fluorescence lifetimes of single Rhodamine 6G molecules on silica surfaces were measured with pulsed laser excitation, time-correlated single photon counting, and near-field scanning optical microscopy (NSOM). The fluorescence lifetime varies with the position of a molecule relative to a near-field probe. Qualitative features of lifetime decreases are consistent with molecular excited state quenching effects near metal surfaces. The technique of NSOM provides a means of altering the environment of a single fluorescent molecule and its decay kinetics in a repeatable fashion.     

Room-temperature detection of a single molecule's absorption by photothermal contrast

So far, single-molecule imaging has predominantly relied on fluorescence detection. We imaged single nonfluorescent azo dye molecules in room-temperature glycerol by the refractive effect of the heat that they release in their environment upon intense illumination. This photothermal technique provides contrast for the absorbing objects only, irrespective of scattering by defects or roughness, with a signal-to-noise ratio of ~10 for a single molecule in an integration time of 300 milliseconds. In the absence of oxygen, virtually no bleaching event was observed, even after more than 10 minutes of illumination. In a solution saturated with oxygen, the average bleaching time was of the order of 1 minute. No blinking was observed in the absorption signal. On the basis of bleaching steps, we obtained an average absorption cross section of 4 angstroms(2) for a single chromophore.     

Vibrationally resolved fluorescence excited with submolecular precision

Tunneling electrons from a scanning tunneling microscope (STM) were used to excite photon emission from individual porphyrin molecules adsorbed on an ultrathin alumina film grown on a NiAl(110) surface. Vibrational features were observed in the light-emission spectra that depended sensitively on the different molecular conformations and corresponding electronic states obtained by scanning tunneling spectroscopy. The high spatial resolution of the STM enabled the demonstration of variations in light-emission spectra from different parts of the molecule. These experiments realize the feasibility of fluorescence spectroscopy with the STM and enable the integration of optical spectroscopy with a nanoprobe for the investigation of single molecules.     

Real-time observation of the vibration of a single adsorbed molecule

The newly developed femtosecond field emission camera was used to observe the time dependence of field emission through a single copper phthalocyanine molecule adsorbed on a tungsten tip. In many of the individual 212-picosecond-long recordings, the field emission was found to oscillate with a frequency between 5 x 10(10) and 20 x 10(10) hertz. The oscillations, which were not observed from a bare tip, are believed to arise from the vibration of a single molecule with respect to the surface. Numerical simulations confirmed the statistical significance of the data.     

Protein hydration in aqueous solution

High-resolution proton nuclear magnetic resonance studies of protein hydration in aqueous solution show that there are two qualitatively different types of hydration sites. A well-defined, small number of water molecules in the interior of the protein are in identical locations in the crystal structure and in solution, and their residence times are in the range from about 10(-2) to 10(-8) second. Hydration of the protein surface in solution is by water molecules with residence times in the subnanosecond range, even when they are located in hydration sites that contain well-ordered water in the x-ray structures of protein single crystals.     

Single molecules observed by near-field scanning optical microscopy

Individual carbocyanine dye molecules in a sub-monolayer spread have been imaged with near-field scanning optical microscopy. Molecules can be repeatedly detected and spatially localized (to approximately lambda/50 where lambda is the wavelength of light) with a sensitivity of at least 0.005 molecules/(Hz)(1/2) and the orientation of each molecular dipole can be determined. This information is exploited to map the electric field distribution in the near-field aperture with molecular spatial resolution.     

Simultaneous fluorescence and Raman scattering from single carbon nanotubes

Single-molecule fluorescence spectroscopy was used to determine the electronic properties of individual single-walled carbon nanotubes. Carbon nanotube structure was determined simultaneously from Raman spectroscopy. Fluorescence spectra from individual nanotubes with identical structures have different emission energies and linewidths that likely arise from defects or the local environment. Unlike most other molecules studied to date, the fluorescence intensity or spectrum from a single nanotube unexpectedly did not fluctuate.     

Illuminating single molecules in condensed matter

Efficient collection and detection of fluorescence coupled with careful minimization of background from impurities and Raman scattering now enable routine optical microscopy and study of single molecules in complex condensed matter environments. This ultimate method for unraveling ensemble averages leads to the observation of new effects and to direct measurements of stochastic fluctuations. Experiments at cryogenic temperatures open new directions in molecular spectroscopy, quantum optics, and solid-state dynamics. Room-temperature investigations apply several techniques (polarization microscopy, single-molecule imaging, emission time dependence, energy transfer, lifetime studies, and the like) to a growing array of biophysical problems where new insight may be gained from direct observations of hidden static and dynamic inhomogeneity.     

Hubble space telescope observations of comet p/shoemaker-levy 9 (1993e)

The Hubble Space Telescope observed the fragmented comet P/Shoemaker-Levy 9 (1993e) (P indicates that it is a periodic comet) on 1 July 1993. Approximately 20 individual nuclei and their comae were observed in images taken with the Planetary Camera. After subtraction of the comae light, the 11 brightest nuclei have magnitudes between approximately 23.7 and 24.8. Assuming that the geometric albedo is 0.04, these magnitudes imply that the nuclear diameters are in the range approximately 2.5 to 4.3 kilometers. If the density of each nucleus is 1 gram per cubic centimeter, the total energy deposited by the impact of these 11 nuclei into Jupiter's atmosphere next July will be approximately 4 x 10(30) ergs ( approximately 10(8) megatons of TNT). This latter number should be regarded as an upper limit because the nuclear magnitudes probably contain a small residual coma contribution. The Faint Object Spectrograph was used to search for fluorescence from OH, which is usually an excellent indicator of cometary activity. No OH emission was detected, and this can be translated into an upper limit on the water production rate of approximately 2 x 10(27) molecules per second.     

Jumping nanodroplets

Flat gold nanostructures on inert substrates like glass or graphite were illuminated by single intensive laser pulses with fluences above the gold melting threshold. The liquid structures produced in this way are far from their equilibrium shape, and a dewetting process sets in. On a time scale of a few nanoseconds, the liquid contracted toward a sphere. During this contraction, the center of mass moved upward, which could lead to detachment of droplets from the surface due to inertia. The resulting velocities were on the order of 10 meters per second for droplets with radii in the range of 100 nanometers.     

Localized dispersal and recruitment in great barrier reef corals: the helix experiment

To examine the problem of how far coral larvae disperse from their natal reef, coral recruitment densities were experimentally determined at distances up to 5 kilometers from a small, relatively isolated platform reef, Helix Reef, on the central Great Barrier Reef for 7 months. High concentrations of recruits, accounting for up to 40 percent of all recruitment, were found downstream of the reef in areas of high water residence time, suggesting that near-field(proximal) circulation has a profound influence on dispersal and recruitment of coral larvae. Coral recruitment declined logarithmically with distance from the reef, decreasing by an order of magnitude at radial distances of only 600 to 1200 meters. On an ecological time scale, advective dispersal of semipassive marine larvae with relatively short planktonic lives(minimally days) may be extensive, but success of recruitment is highly limited. Through evolutionary time, sufficient dispersal occurs to ensure gene flow to reef tracts hundreds or possibly thousands of kilometers apart. In the short term, however, coral reefs appear to be primarily self-seeded with respect to coral larvae.     

太行山南麓刺槐人工林土壤呼吸与土壤温度间的滞后关系

目的土壤呼吸是陆地生态系统碳循环的一个重要组成部分。本研究在年尺度和日尺度上分析刺槐人工林土壤呼吸与各土层温度间的温度敏感系数（Q₁₀）的差异,并探讨土壤呼吸与各土层温度间的时间滞后。方法利用土壤呼吸自动观测系统Li-8150对太行山南麓50年生刺槐林进行定位观测。结果在年尺度上,刺槐人工林土壤呼吸与各土层温度在时间上的滞后不明显,且呈现显著的指数关系（P < 0.001）。Q₁₀在2.23 ~ 2.53之间变动,且Q₁₀随土壤温度测量深度的增加而略微增大,指数模型拟合系数（R2）随土壤温度测量深度的增加而略微减小。土壤呼吸与各土层温度不需要相位校准。在日尺度上,土壤呼吸与各层温度存在时间上的滞后,拟合系数R2较小,获得Q₁₀并不准确。土壤呼吸与各土层温度需要进行相位校准,校准后的土壤呼吸与土壤温度间的拟合系数R2增大,获得的Q₁₀更准确,并且Q₁₀值随土壤温度测量深度增加而增加,但是深层（> 20 cm）的Q₁₀过大并不符合生物学规律。在土壤浅层（< 20 cm）,Q₁₀随深度增加与土层温度变化幅度有关。结论在年尺度上,各土层Q₁₀值相差不大,都能较理想反映全年刺槐林土壤呼吸与温度的关系。在日尺度上,本研究推荐使用浅层的Q₁₀来反映日间刺槐林土壤呼吸与土壤温度关系。      

An expanded palette of genetically encoded Ca²⁺ indicators

Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.     

Breaking the diffraction barrier: optical microscopy on a nanometric scale

In near-field scanning optical microscopy, a light source or detector with dimensions less than the wavelength (lambda) is placed in close proximity (lambda/50) to a sample to generate images with resolution better than the diffraction limit. A near-field probe has been developed that yields a resolution of approximately 12 nm ( approximately lambda/43) and signals approximately 10(4)- to 10(6)-fold larger than those reported previously. In addition, image contrast is demonstrated to be highly polarization dependent. With these probes, near-field microscopy appears poised to fulfill its promise by combining the power of optical characterization methods with nanometric spatial resolution.     

Subcellular sources of luminescence in Noctiluca

The perivacuolar cytoplasm of Noctiluca miliaris contains approximately 10(4) microsources of luminescence, with dimensions of 0.5 to 1.5 microns, which exhibit marked fluorescence with ultraviolet excitation. Local invasion by an action potential elicits light emission (microflashes) from these sources with a coupling latency of about 2 milliseconds. Magnitudes of the microflash vary directly with the dimensions of the source. Time courses of the microflash resemble that of the macroflash emitted by the whole cell but have somewhat shorter time constants. The small discrepancy induration between micro- and macroflash can be explained by the 5- to 10-millisecond asynchrony of microsource triggering that results from the conduction time of the action potential. Reversible gradations in amplitude of the macroflash, as from potentiation or fatigue, result from parallel summation of graded changes in microflash intensity. Thus the macroflash gives a reasonably true picture of the subcellular kinetics of luminescence.     

光纤化学传感荧光法测定维生素B2片剂含量的研究

[目的]利用光纤化学传感荧光法,准确测定维生素B2片剂的含量.[方法]利用光纤化学传感荧光法检测系统,将维生素B2片溶解于水溶液中,测定其荧光光谱,定量分析其含量.[结果]该方法维生素B2最大发射波长为533 nm,质量浓度在3.2×10-5～8×10-4 mg/ml与荧光强度呈良好线性关系,检测线为1.55×10-8 mg/ml,日内精密度为0.19％,日间精密度为1.2％,回收率为97.9％ ～105.1％.[结论]采取该方法测定维生素B2药片含量的准确度高,检测线低,结果令人满意.     

胶束增溶荧光体系测定微量铁的研究

在HAc-NaAc缓冲介质（pH值5.00）中,发现溶液中的铁（Ⅲ）能与茜素黄-罗丹明B反应生成稳定的络合物且使其内源性荧光明显猝灭。据此,建立铁（Ⅲ）-茜素黄-罗丹明B-吐温80体系荧光猝灭体系法测定铁的新方法。该体系的最大激发波长λex=365nm,最大发射波长λem=585nm,铁（Ⅲ）的浓度在1.20×10^-7～1.80×10^-5mol/L时与其荧光强度差值△F呈良好的线性关系。方法的检出限为8.02×10^-8mol/L,相对标准偏差为1.02%,回收率为97.64%～102.39%。采用该方法测定水和茶叶中的铁,结果令人满意。     

Nanoscale imaging of buried structures via scanning near-field ultrasound holography

A nondestructive imaging method, scanning near-field ultrasound holography (SNFUH), has been developed that provides depth information as well as spatial resolution at the 10- to 100-nanometer scale. In SNFUH, the phase and amplitude of the scattered specimen ultrasound wave, reflected in perturbation to the surface acoustic standing wave, are mapped with a scanning probe microscopy platform to provide nanoscale-resolution images of the internal substructure of diverse materials. We have used SNFUH to image buried nanostructures, to perform subsurface metrology in microelectronic structures, and to image malaria parasites in red blood cells.     

冷冻胁迫下方竹抗氧化酶活性和叶绿素荧光特性

以1年生盆栽实生方竹为材料,人工模拟冷害(4℃)、冻害(-5,-10,-20℃)和短时间(15,30,60 min)胁迫,测定抗氧化酶(CAT,POD,SOD)活性的变化,同时通过Imaging-PAM观测叶绿素荧光参数的变化,探讨方竹抵抗冷冻胁迫的机制。结果表明,冷害和冻害胁迫均可诱导方竹体内防御体系发挥作用,方竹叶片的相关抗氧化酶活性均表现出先升高后降低再升高的趋势,其中CAT和POD活性对低温胁迫的应答较为迟缓,而SOD活性对冷害和冻害胁迫的反应更灵敏。在冷害与冻害胁迫下,方竹叶片的叶绿素荧光参数Fv/Fm、Y(Ⅱ)、qP、ETR均呈现先降低后升高的趋势;与4℃低温的冷害胁迫相比较,冻害胁迫对光合作用的损伤更严重,其中在-20℃处理下,ETR、Y(Ⅱ)、qP皆为0,表明此时方竹已失去光合能力。     

以铕离子为荧光探针测定痕量环丙沙星

本文研究了环丙沙星( CIP)同铕离子形成的配合物体系的荧光光谱特性及实验条件对荧光强度的影响.在pH值为8.02的Tris-HCl条件下,Eu3+和环丙沙星(CIP)能形成配合物发生分子内能量转移,发射铕离子的特征荧光,其最大激发和发射波长为265nm和615nm.加入阴离子表面活性剂十二烷基硫酸钠(SDS)后,可极大地增强体系的荧光强度,据此建立了一种以铕离子为荧光探针分析痕量环丙沙星含量的新方法,方法的线性范围为2.46×10-7mol· dm-3～9.84×10-5 mol·dm-3,相关系数r=0.9965,检出限为1.92×10-7mo 1·dmn-3.该法用于环丙沙星片剂和针剂的测定,回收率在98％～103％之间,结果令人满意.