荧光光谱用于光动力疗法中光敏剂光漂白特性研究

应用荧光光谱技术研究溶液中血卟啉单甲醚(HMME)的光漂白与光产物生成。以532 nm倍频Nd∶YAG激光器照射样品,功率密度为100 mW·cm-2,以光学多通道分析仪(OMA)采集荧光光谱。照光过程与荧光光谱采集同步进行。通过构建基本光谱与最小二乘拟合,由单条实测光谱中分解求得HMME荧光(613 nm)、光产物荧光(639 nm)及自体荧光的强度。HMME初始浓度不超过10 μg·mL-1时符合荧光-浓度线性函数关系。对照光过程的荧光光谱监测同时观察到HMME漂白、光产物生成与漂白,以及样品光学特性变化引起的自体荧光强度起伏。光产物漂白后的二次产物引起样品光学特性显著改变。所建立的荧光光谱探测系统与光谱分析方法可满足光敏剂漂白特性体外研究的需要,并为光动力治疗的剂量学在体监测提供有效研究方法。     

Resolution of <Emphasis Type="BoldItalic">λ</Emphasis> /10 in fluorescence microscopy using fast single molecule photo-switching

We demonstrate nanoscale resolution in far-field optical microscopy based on photo-switching of molecules. By enabling, recording and disabling fluorescence from individual labels sequentially, the detection volume is reduced to the size of a single molecule and the diffraction limit is broken. Images of nanostructures milled into a coverslip and tagged by fluorescent proteins could be recorded at 50 nm resolution. Due to the fast and asynchronous image acquisition protocol used in these experiments, we were able to reduce acquisition times to ∼2.5 min, which is two orders of magnitude lower than in previous implementations. PACS 81.07.-b; 42.30.-d; 87.64.-t     

A single molecule as a probe of optical intensity distribution

Single terrylene molecules embedded in microscopic p-terphenyl crystals are identified with the technique of fluorescence excitation spectroscopy. By use of the architecture of a scanning-probe microscope at T = 1.4 K , a single molecule is scanned through an excitation laser beam while the fluorescence signal is recorded. In this manner we have mapped the intensity distribution in a one-dimensional optical standing wave, demonstrating the potential of a single molecule as a nanometric probe. We discuss future experiments aimed at combining the high spatial and spectral sensitivity of a single molecule.     

Simultaneous time- and wavelength-resolved fluorescence spectroscopy for near real-time tissue diagnosis

A novel fiber-optic-based method for simultaneous time- and wavelength-resolved fluorescence spectroscopy for the rapid diagnosis of diseased tissue is demonstrated. By combining multiple bandpass and dichroic filters (405/40, 460/50, and 550/50) with different lengths of optical fiber (1, 10, and 19 m) acting as an optical delay this system enables the near real-time acquisition and characterization of time-resolved fluorescence spectra using a single detector and excitation input. The recording of multiple fluorescence response pulses at selected wavelengths can be completed in hundreds of nanoseconds, which provides the capability of a real-time characterization of biological systems.     

泵浦蓝宝石光纤荧光温度传感器

采用激光加热基座法制备端部Cr3＋离子掺杂的蓝宝石单晶光纤,得到一体型蓝宝石单晶光纤荧光温度传感头.对所制备的荧光温度传感头的荧光温度特性进行了实验研究.结果表明,随着温度升高Cr3＋∶Al2O3单晶光纤荧光寿命单调下降,从温度为0 ℃的4.0 ms下降到450 ℃的0.2 ms.利用所制备的荧光温度传感头,用波长405 nm紫色LED作为泵浦光源,采用相关检测技术在线实时测量荧光寿命,研制成测温范围0 ℃～450 ℃一体型蓝宝石光纤荧光温度传感器.     

Near-field scanning optical microscopy of single molecules by femtosecond two-photon excitation

We present a demonstration of near-field scanning optical microscopy of single molecules based on ultrafast two-photon-induced fluorescence. Measurements were performed by use of 100-fs pulses at 800 nm from a Ti:sapphire laser to excite the two-photon transition in Rhodamine B molecules. Although near-field probes are normally metal coated to achieve superresolution, we used uncoated tips to achieve sufficiently high optical powers to generate acceptable fluorescence emission rates. Images of single molecules demonstrate a resolution of ~175nm(< lambda/4) on a topographically smooth surface, which surpasses the apparent lambda/2 resolution limit for uncoated tips operating in the linear response regime.     

Investigation of the Doughnut Effect in Cy3-Labeled Protein Microarrays using Scanning Near-Field Optical Microscope

We demonstrate the fluorescence mapping of protein microarrays by the technique of scanning near-field optical microscopy (SNOM) and confocal microscopy. Micron sized spots (300 μm) of human Immunoglobulin G (hIgG) protein with and without a Cy3 dye labeling have been fabricated on glass substrates by an immobilization method which makes use of calixcrown derivatives termed Prolinker. We have also tried to probe into the well-known “doughnut effect” observed in fluorescence images of proteins using the SNOM technique. The topographic and fluorescence SNOM images revealed that the number of proteins at the boundary of the spot were more than at the center in the case of the microarray spot which showed brighter luminescence at the edge than at the center in the confocal image.     

High-resolution imaging of single fluorescent molecules with the optical near-field of a metal tip

We show that a concentration of light at a metal tip allows near-field optical imaging of single fluorescent dye molecules at very high resolution, despite strong quenching effects. Details as small as 10 nm were observed in the fluorescence patterns of single Cy-3 dyes bound to the termini of DNA. Data evaluation by model fitting determines the positions of the dyes to an accuracy even better than 1 nm and also yields their 3D orientation. The metal tip simultaneously provides high-resolution topographic imaging complementing the optical signal for a detailed surface examination.     

Spin coherence during optical excitation of a single nitrogen-vacancy center in diamond

We examine the quantum spin state of a single nitrogen-vacancy (NV) center in diamond at room temperature as it makes a transition from the orbital ground state (GS) to the orbital excited state (ES) during nonresonant optical excitation. While the fluorescence readout of NV-center spins relies on conservation of the longitudinal spin projection during optical excitation, the question of quantum phase preservation has not been examined. Using Ramsey measurements and quantum process tomography of the optical excitation process, we measure a trace fidelity of F=0.87±0.03, which includes ES spin dephasing during measurement. Extrapolation to the moment of optical excitation yields F≈0.95. This result provides insight into the interaction between spin coherence and nonresonant optical absorption through a vibronic sideband.     

Effect of Temperature on Re-entrant Condensation of Globular Protein in Presence of Tri-valent Ions

Globular proteins play several essential roles in functioning different mechanisms of the living organisms, and the stability of such protein molecules in an aqueous solution is strongly affected by multivalent ions. In this article, we have systematically studied the effect of temperature (between 5 and 25ºC) on the re-entrant condensation behaviour of bovine serum albumin (BSA) in the presence of trivalent ions, Yttrium (Y³⁺), and Lanthanum (La³⁺). The effect of temperature is explained considering the optical properties of the protein, i.e., from the optical absorption and emission behaviours. The absorption in the visible region and the fluorescence emission of BSA becomes maximum at the lowest temperature. The decrement of mobility at lower temperature is responsible for fluorescence enhancement. Moreover, the activation energy of the turbid or viscus phase of the BSA protein under re-entrant condensation is enhanced in comparison with the transparent phase and the corresponding energy value is estimated from the fluorescence study.

     

Multiplexed time-correlated single-photon counting

We review the technique of multiplexed time-correlated single-photon counting whereby multiple fluorescence decay curves are recorded in parallel by statistically time-sharing a single time-to-amplitude converter. Application of the multiplexing technique to measuring the fluorescence lifetime topography of a self-absorbing sample is demonstrated. Further possibilities are discussed for multiplexed optical fiber sensor networks with built-in intelligence for detecting and discriminating between different metal ions in solution.     

Modification of single molecule fluorescence by a scanning probe

We examine the optical near-field interaction between different types of scanning tips and single oriented fluorescent molecules. We demonstrate the influence of a tip on the excitation intensity as well as on the integrated fluorescence signal, the excited state lifetime, and the angular emission of single molecules. By using a standard model describing the radiation of an oscillating dipole close to a nanosphere or a flat interface, we interpret our observations and describe some central criteria for obtaining fluorescence enhancement or quenching. PACS 33.80.-b; 07.79.Fc; 78.90.+t     

Plasmonic-enhanced two-photon fluorescence with single gold nanoshell

Single gold nanoshell with mutilpolar plasmon resonances is proposed to enhance two-photon fluorescence efficiently.The single emitter single nanoshell configuration is studied systematically by employing the finite-difference time-domain method.The emitter located inside or outside the nanoshell at various positions leads to a significantly different enhancement effect.The fluorescent emitter placed outside the nanoshell can achieve large fluorescence intensity given that both the position and orientation of the emission dipole are optimally controlled.In contrast,for the case of the emitter placed inside the nanoshell,it can experience substantial two-photon fluorescence enhancement without strict requirements upon the position and dipole orientations.Metallic nanoshell encapsulating many fluorescent emitters should be a promising nanocomposite configuration for bright two-photon fluorescence label.The results provide a comprehensive understanding about the plasmonic-enhanced two-photon fluorescence behaviors,and the nanocomposite configuration has great potential for optical detecting,imaging and sensing in biological applications.     

单分子的荧光特性及其在生物学上的应用

近年来,单分子探测在许多学科领域的研究取得了显著的进展。它为科学家提供了一种新的手段来研究这些领域的前沿课题。光学和光谱技术是单分子探测最常用的方法之一。单个分子的荧光强度的涨落及其荧光的偏振特性是单分子荧光的重要特征,在单分子探测的广泛应用中,人们正是利用这种单个分子的重要特征来研究和推测生物大分子的结构和功能。文章简要介绍了单分子的荧光特点、探测方法及其在生物学中的应用。     

Single-molecule detection using continuous wave excitation of two-photon fluorescence

Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.     

Detection of Single Quantum Dots in Model Systems with Sheet Illumination Microscopy

Single molecule detection and tracking provides at times the only possible method to observe the interactions of low numbers of biomolecules, inlcuding DNA, receptors and signal mediating proteins in living systems. However, most existing imaging methods do not enable both high sensitivity and non-invasive imaging of large specimens. In this study we report a new setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with the resolution of confocal microscopy and the optical penetration beyond 300 μm. We detect and report our instrumental figures of merit, control values of fluorescence properties of single nano crystals in comparison to both standard widefield configurations, and also values of nanocrystals in multicellular “fruiting bodies” of Dictyostelium, an excellent control as a model developmental system. In the Dictyostelium , we also report some of our first tracking of single nanocrystals with SPIM. The new SPIM setup represents a new technique, which enables fast single molecule imaging and tracking in living systems.     

Fluctuations in the absorption coefficient of a single molecule at different signal acquisition times

The relation between the absorption coefficient of a single molecule calculated within the framework of a dynamical theory and the fluctuations in the absorption coefficient measured in experiments on scanning the laser frequency is analyzed in relation to the acquisition time of fluorescence photons. The analysis is performed for the case where the optical band of an impurity center consists of two well-resolved lines. A method of processing the fluctuating fluorescence is proposed that makes it possible to obtain the averaged absorption coefficients, which can be compared with the coefficients calculated theoretically.     

Optical amplification using raman transitions between spin-singlet and spin-triplet states of a pair of coupled in-GaAs quantum dots

We report the observation of steady-state optical amplification in Raman transitions between the lowest-energy spin states of a single quantum-dot molecule. Absorption and resonance fluorescence experiments demonstrate that the entangled two-electron singlet and triplet states have electric-dipole coupling to a common optically excited state. Fast spin relaxation ensures optical gain on the triplet transition when the singlet transition is driven resonantly. By embedding the quantum-dot molecule in a cavity of modest quality factor, a solid-state single-emitter laser can be realized.     

Toward oxygen binding curves of single respiratory proteins

Oxygen binding curves of single molecules promise to discriminate between different models describing cooperativity because load distributions are accessible. Individual tarantula hemocyanins could be detected by fluorescence correlation spectroscopy using intrinsic tryptophan fluorescence as sensor of bound oxygen. However, imaging of immobilized proteins was not possible due to fast photo-bleaching. It is shown that tetra-methyl-carboxy-rhodamine (TAMRA), commonly used as a fluorescence label in single-molecule spectroscopy, can also be applied to monitor bound oxygen. The dye's fluorescence is quenched due to F?rster energy transfer to the oxygenated active sites of hemocyanin.     

Quantum dot labeling based on near-field optical imaging of CD44 molecules

The lateral organization of membrane proteins and lipids domains has a direct impact on many cellular processes, but generally these domains are too small to be resolved by diffraction-limited resolution of fluorescence microscopy. Here, we use quantum dot (QD) labeling based on near-field optical imaging, to study the nanoscale organization of hyaluronan receptor CD44 molecules of fixed mesenchymal stem cells (MSCs) in air, with a optical resolution down to 50 nm. The photostability and high luminance of QD evidently improve the signal-to-noise ratio and reproducibility of near-field optical data. Importantly, the blinking-intensity analysis was proposed to identify single QD, providing a calibration to relate intensity to numbers of antibody for the first time. Additionally, the fluorescence-topographic imaging enables us to investigate the topographic location pattern. Our results demonstrate that CD44 molecules on MSCs are enriched into nanosized domain and they predominantly locate on the peak of the membrane protrusions, which may contribute to clarify the underlying mechanism of functions ascribed to these molecules.