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.     

Epicocconone, A New Cell-Permeable Long Stokes' Shift Fluorescent Stain for Live Cell Imaging and Multiplexing

Epicocconone is a heterocyclic natural product from the fungus Epicoccum nigrum that fluoresces weakly in the green (520 nm). However, cells exposed to epicocconone rapidly absorb the dye and become bright orange fluorescent because the natural product reacts reversibly with proteins. The orange fluorescence is enhanced in lipophilic environments, allowing the visualization of membranous organelles and lipid rafts but does not stain oligonucleotides. As the unconjugated dye has no orange fluorescence, there is no need to wash out the excess fluorophore. Epicocconone is a neutral, non-toxic, small molecule that appears to diffuse readily into live of fixed cells without the need for permeabilization. These features enable the real-time imaging of live cells and the study of organelle movements. Cells stained with epicocconone are excitable by common lasers (UV, 405, 488, and 532 nm) and its long Stokes' shift allows multiplexing applications with more common short Stokes' fluorophores using a single light source.     

Picosecond decay kinetics of CdS luminescence studied by a streak camera

Using a picosecond laser and a streak camera we have observed the time dependence of the luminescence intensity of free excitons, bound excitons, and excitonic molecules in CdS. The observed kinetics show that the P band is due to bimolecular emission from free excitons and that bound excitons are generated from free excitons through monomelecular process and excitonic molecule through bimolecular process.     

Investigation of Abnormal Long‐Wavelength Fluorescence Emissions Occurring in Binary Organic Nanoparticle Films

In some fluorophores with planar groups, a long‐wavelength emission band different from their native one can sometimes be observed. The main cause of this long‐wavelength band is excimer formation. It is generally accepted that once condensed in the solid state, whether fluorophores can exhibit excimer emission or not depends only on their molecular structure and packing. However, here it is shown that there are exceptions when fluorophores are present in nanoparticles (NPs), where excimer emission can be affected by the environment surrounding the NPs, even in the solid state. It is found that in some binary NP films consisting of fluorophore NPs and other NPs, unusual long‐wavelength bands ascribed to excimer emission can be activated, even though these bands are absent from the photoluminescence spectra of the pure fluorophore NP films. This finding is beneficial to better understand and control excimer emissions. In addition, such a binary NP system provides an ideal platform to investigate the interplay between two fluorophores, because it keeps them effectively separated while maintaining suitable spatial distances for exciton migration and dipole–dipole interactions. This work also provides evidence for the long‐debated origin of the green emission band (g‐band) of fluorene‐based fluorophores.     

Boronic Acid Based Modular Fluorescent Sensors for Glucose

Modular photoinduced electron transfer (PET) sensors bearing two phenylboronic acid groups, one or two fluorophores: pyrene(a), phenanthrene(b), anthracene(c), 1-naphthalene(d), 2-naphthalene(e) and alkylene linkers, from trimethylene(3) to octamethylene(8), have been evaluated. Systems with a single pyrene fluorophore 34a, 35a and 36a bind the strongest with D-glucose (36a also binds well with D-melibiose). Whilst 37a and 38a bind the strongest with D-galactose. Changing the fluorophore, also, influences the binding, 36a, 36b and 36c are D-glucose selective, whilst 36d and 36e are D-galactose selective. Systems with two fluorophores 36a-a and 36a-b show an overall decrease in binding efficiency. Energy transfer in 36a-b results in enhanced sensitivity and selectivity towards D-glucose.     

Picosescond fluorescence lifetime standards for frequency- and time-domain fluorescence

We characterized a series of dimethylamino-stilbene derivatives as standards for time-domain and frequency-domain lifetime measurements. The substances have reasonable quantum yields, are soluble in solvents available with a high purity, and do not show significant sensitivity to oxygen quenching. All the fluorophores displayed single exponential intensity decays, as characterized by frequency-domain measurements to 10 GHz. The decay times vary from 880 to 57 ps, depending on structure, solvent, and temperature, which is a useful range for modern picosecond time-domain or gigahertz frequency-domain instruments. These fluorophores may be used either to test an instrument or as reference compounds to eliminate color effects. We also characterized two-fluorophore mixtures, with the decay times spaced twofold (150 and 300 ps), with varying proportions. These mixtures are useful for testing the resolution of other time- and frequency-domain instrumentation. The excitation wavelength ranges from 260 to 430 nm, and the emission from 350 to 550 nm. The decay times are independent of the excitation and emission wavelengths.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术,对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究,尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明,利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团,利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具,对眼底细胞随年龄增长的衰老机理的研究具有重要的意义. 关键词： 双光子激发荧光 荧光寿命成像 视网膜色素上皮层     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术，对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究，尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明，利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团，利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具，对眼底细胞随年龄增长的衰老机理的研究具有重要的意义.     

拟威布尔分布密度函数在荧光寿命成像数据分析中的应用

荧光寿命法成像技术(FLIM)是一种非常有效、功能强大且能用来分析复杂生物组织和细胞分子的成像技术。传统的荧光寿命成像的数据分析,按某些具有不同寿命、离散的单参量指数模型来描述荧光衰减过程。在生物组织这样既复杂又不均匀的样品中,虽然多参量指数模型能提供比单参量指数模型对实验数据更好的拟合效果,但是离散多参量的假定往往是随意的。提出了拟威布尔分布密度函数可能是生物荧光分子团衰减动力过程的真实再现,并且通过计算证明,对于某些生化感兴趣的荧光分子团的多槽基面效价测定样品的数据,相对于单参量指数与多参量指数衰减函数有更好的一致性。同时讨论了将该荧光衰减模型应用于荧光寿命成像的前景。     

Utility and Considerations of Donor–Donor Energy Migration as a Fluorescence Method for Exploring Protein Structure-Function

This review aims at surveying the use of electronic energy transport between chemically identical fluorophores (i.e. donors) in studies of various protein systems. Applications of intra- and interprotein energy migration are presented that make use of polarised steady-state and time-resolved fluorescence spectroscopic techniques. The donor-donor energy migration (DDEM) and the partial donor-donor energy migration (PDDEM) models for calculating distances between donor groups are exposed together with the most recent development of an extended Forster theory (EFT). Synthetic fluorescence depolarisation data that mimic time-correlated single photon counting experiments were generated using the EFT, and then further re-analysed by the different models. The results obtained were compared with the known parameters used to generate EFT data. Aspects on how to adopt the EFT in the analyses of time-correlated single photon counting experiments are also presented, as well as future aspects on using energy migration for examining protein structure.     

多色双光子激发荧光显微技术实验研究

双光子激发荧光(two-photon excited fluorescence, TPEF)显微是一种非线性光学显微技术, 具有高的时间分辨率和空间分辨率、高的信噪比和固有的三维层析分辨能力等优点. 传统的TPEF显微一般采用波长可调谐的超短脉冲激光器作为光源. 在实际应用中, 利用TPEF显微技术研究含有多种荧光团或未知成分的待测样品, 往往需要多次改变激发光的波长以获得对各种荧光团的最佳激发. 为了同时获取不同荧光团的荧光信号, 利用超连续谱激光光源实现了多色TPEF显微成像, 实验中无需调节波长, 能够同时获得具有两种不同发射波长的荧光标记的铃兰根茎切片样品的TPEF图像. 实验结果表明, 与传统的TPEF显微相比, 该方法能够同时获取含有多种荧光团的待测样品的高对比度TPEF图像, 具有系统结构简单、操作简便、信息量大等优点, 在生物医学和材料科学等领域具有广阔的应用前景.     

Rapid Clearance of IR783 and Methyl-β-cyclodextrin Complex for Improved Tumor Imaging

Near-infrared (NIR) fluorescence cancer imaging with targeted NIR fluorophores holds considerable promise for accurate detection and cancer diagnosis. Among the various NIR heptamethine cyanine dyes reported previously, IR783 as a single small molecule has been widely used for tumor-targeted imaging without the additional conjugation of targeting moieties. Despite the potential advantages of IR783, the major problems, such as its non-specific uptake in normal tissues/organs and slow clearance, remain to be solved. A key determinant of sensitivity and detectability in tumor imaging is the improvement of the tumor-to-background ratio (TBR). Herein, a simple and effective supramolecular complex self-assembled from IR783 and methyl-β-cyclodextrin is developed to improve tumor imaging accompanied by rapid clearance from the body. The IR783-cyclodextrin complex allowed for rapid whole body biodistribution, which remarkably reduced non-specific background uptake, and thus increased the TBR value within 24 h post-injection. Therefore, this strategy is applicable in combination with many different types of carbocyanine dyes for improved tumor imaging.     

Single molecule lifetime fluctuations reveal segmental dynamics in polymers

We present a single molecule fluorescence study that allows one to probe the nanoscale segmental dynamics in amorphous polymer matrices. By recording single molecular lifetime trajectories of embedded fluorophores, peculiar excursions towards longer lifetimes are observed. The asymmetric response is shown to reflect variations in the photonic mode density as a result of the local density fluctuations of the surrounding polymer. We determine the number of polymer segments involved in a local segmental rearrangement volume around the probe. A common decrease of the number of segments with temperature is found for both investigated polymers, poly(styrene) and poly(isobutylmethacrylate). Our novel approach will prove powerful for the understanding of the nanoscale rearrangements in functional polymers.     

Syntheses of Novel Dipyrrylmethene-BF2 Dyes and Their Performance as Labels in Two-Photon Excited Fluoroimmunoassay

Two-photon excitation of fluorescence (TPE) has been found a powerful tool in the field of microscopy imaging and recently also in the field of bioanalytics. The recently introduced bioaffinity assay technology, ArcDia TPX, enables separation-free ultra-sensitive immunoassays from microvolumes. This assay technique is based on the use of microspheres as a solid reaction carriers and two-photon excited fluorescence detection. In the ArcDia TPX-technology, the individual microparticles are observed and the number of bound biomolecules on the microparticle surface is quantified by two-photon excited fluorescence. Here we present synthesis and use of a novel dipyrrylmethene-BF2 fluorophore that has been designed to be used as label in ArcDia TPX assay technique. The absorption and emission wavelengths of the label are tuned to allow excitation with a 1064 nm microchip laser. The label contains two-carboxylic residues, one of which is activated as N-hydroxysuccinimide ester to enable labeling of amino residues of biomolecules. The other carboxylic group is in free form to increase solubility in aqueous solutions. This new fluorescent label is tested in a separation-free immunoassay using ArcDia TPX assay technique. The performance of the new label is compared to that of one of the brightest fluorophores available, R-phycoerythrin (RPE). According to the results, the dipyrrylmethene-BF2 label provides significantly better signal-to-background ratio, leading to higher assay sensitivity and broader dynamic range compared to that of RPE. Good solubility to aqueous solutions and high fluorescence quantum efficiency, suggests the dipyrrylmethene-BF2 label is applicable also in other fluorescence-based applications.     

Single Molecule Fluorescence Imaging and Its Application to the Study of DNA Condensation

Single molecule fluorescence imaging incorporated with optical tweezers and a laminar flow cell has been used to monitor the kinetic process of DNA condensation induced by spermidine. It was found that at least two steps were involved in the condensation process of the hydrodynamically-stretched linear DNA; a lag period followed by a rapid collapse of DNA. The lag time increased with the flow speed and the collapse time remained short within the range of the flow speed studied. The effect of salt concentration on the condensation process was examined, and the results suggest that the longer lag time observed in the higher salt buffer probably results from the displacement of bound cations and rearrangement of spermidine on the DNA. The flow-speed dependence of the lag time suggests that a nucleation event at the free end of the DNA, i.e. formation of a loop, may play a vital role in the kinetic process of condensation.     

Asymmetrical Distribution of Intrinsic Fluorescence Lifetimes in Proteins

Dynamic fluorescence measurements of proteins in solution are often interpreted in terms of continuous distributions of lifetimes, which reflect the intrinsic structural heterogeneity of these systems. In several cases a single Gaussian or Lorentzian symmetric distribution has been used to fit the data. In this paper we describe a new nonsymmetric Lorentzian function which contains three free parameters (the center, the left, and the right widths) like the double-discrete exponential model (the two lifetimes and one preexponential factor). Simulated data in the frequency domain have been used to compare the fits obtained with these different approaches, introducing a new parameter, , which quantitatively measures the asymmetry of the distribution or the ratio of the two preexponential factors, in the continuous and discrete models, respectively. Real measurements of a mixture of independent fluorophores, as well as of protein fluorescence decays, have also been performed and analyzed in terms of the new asymmetric function. The data have also been fitted with traditional discrete methods (such as the two- and the three-exponential decay) and with another asymmetric function, namely, the skewed Gaussian distribution.     

Metal-Enhanced Fluorescence from Plastic Substrates

We report the first findings of Metal-Enhanced Fluorescence (MEF) from modified plastic substrates. In the past several years our laboratories have reported the favorable effects of fluorophores in close proximity to silver nanoparticles. These effects include, enhanced fluorescence intensities, (increased detectability), and reduced lifetimes, (enhanced fluorophore photostability). All of these reports have featured silver nanostructures and fluorophores which have been immobilized onto clean glass or quartz surfaces. In this report we show how plastic surfaces can be modified to obtain surface functionality, which in turn allows for silver deposition and therefore metal-enhanced fluorescence of fluorophores positioned above the silver using a protein spacer. Our findings show that plastic substrates are ideal surfaces for metal-enhanced phenomena, producing similar enhancements as compared to clean glass surfaces. Subsequently, we speculate that plastic substrates for MEF will find common place, as compared to the more expensive and less versatile traditional silica based supports.     

单分子科学进展

综述了新兴边缘学科－－单分子科学的进展。对单分子科学的基本实验技术即扫描探针显微术、荧光技术和光镊技术进行了介绍。结合众多的实例（如：对单原子的直接操纵、直接测量化学键强度、在DNA链上“拔河”、用单个C60分子作放大器等）评述了单分子科学方法在各学科领域的广泛应用,以及单分子科学对它们产生的深远影响。最后对单分子科学的发展前景进行了展望。     

Obviously Enhanced Fluorescent Signal of Core-Shell Nanostructures through Simultaneous Regulation of Spectral Overlap and Shell Thickness for Imaging and Photothermal Therapy of Ovarian Cancer Cells

In this research, by simultaneously regulating the two major factors affecting the plasmonic enhanced fluorescence (PEF), spectral overlap and the distance between the fluororophores and the noble metal nanoparticles, a significantly enhanced fluorescent signal is achieved. Core-shell nanostructures composed of aspect ratio (AR) adjustable gold nanorods (GNRs) and various thickness of SiO₂ are prepared and the decorated fluorophores are realized optimized PEF. A typical stimuli-responsive conjugated polymer, polydiacetylene (PDA), and a near-infrared (NIR) dye Cy5.5 are selected as fluorophores and their fluorescent signal are enhanced 7.26 and 4.41 times, respectively. Based on the optimized optical properties, a multifunctional antibody modified Mab-Cy5.5-GNRs@SiO₂ is successfully demonstrated the targeting, imaging, and photothermal therapy (PTT) effects on SKOV-3 ovarian cancer cells.     

Oxygen-dependent photochemistry of fluorescent dyes studied at the single molecule level

We perform wide-field microscopy to investigate the photobleaching of organic fluorophores embedded in the polymeric host PMMA. Our experimental arrangement facilitates the comparison between the ensemble and single molecule data. We characterize the photostability of dye molecules of various families by measuring the ‘bleaching number’, defined as the average number of photons a molecule emits until photobleaching occurs. In particular, we have analysed the dependence of the bleaching number on the presence of oxygen. Surprisingly, we find an improvement of photostability in the presence of oxygen for ionic dyes (DiI, TMR, Rh6G, Alexa 546), suggesting that oxygen quenches the photoactive triplet state, but it only indirectly contributes to photochemistry. In contrast, we observe that photobleaching of the aromatic hydrocarbon is strongly enhanced by oxygen.