Dual‐Mode Superresolution Imaging Using Charge Transfer Dynamics in Semiconducting Polymer Dots

In a conjugated polymer‐based single‐particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single‐particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin‐coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.     

High-Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super-Resolution Imaging

The spatial resolution of single-molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single-particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4–9 frame binning, we achieved a 2–3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.     

Fluorescence Nanoscopy with Optical Sectioning by Two‐Photon Induced Molecular Switching using Continuous‐Wave Lasers

During the last decade far‐field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous‐wave laser beam. This bright marker enables strict control of on/off switching and provides single‐molecule localization precision down to 15 nm in the focal plane. Two‐photon induced nonlinear photoswitching of this marker with continuous‐wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost‐effective fluorescence nanoscopy with noninvasive optical sectioning.     

Nanoscale vibrational analysis of single-walled carbon nanotubes

We use near-field Raman imaging and spectroscopy to study localized vibrational modes along individual, single-walled carbon nanotubes (SWNTs) with a spatial resolution of 10-20 nm. Our approach relies on the enhanced field near a laser-irradiated gold tip which acts as the Raman excitation source. We find that for arc-discharge SWNTs, both the radial breathing mode (RBM) and intermediate frequency mode (IFM) are highly localized. We attribute such localization to local changes in the tube structure (n, m). In comparison, we observe no such localization of the Raman active modes in SWNTs grown by chemical vapor deposition (CVD). The direct comparison between arc-discharge and CVD-grown tubes allows us to rule out any artifacts induced by the supporting substrate.     

Absorbance detector for high-performance liquid chromatography based on light-emitting diodes for the deep-ultraviolet range

A HPLC-detector has been designed which employs light-emitting diodes in the deep-UV-range below 300 nm as wavelength specific radiation sources and special UV-photodiodes for measuring the signal. A monochromator is therefore not needed. The design features a beam splitter and a reference photodiode, precision mechanics for adjustment of the light beams and electronics for stabilization of the LED-current. The processing of the photodiode currents is carried out with a high performance log-ratio amplifier which allows direct absorbance measurements. The optical and electronic performance of the detector was characterised and high precision over several absorbance units was obtained. Testing of analytical separation methods in isocratic as well as gradient modes employing UV-detection at 255 and 280 nm showed a very similar performance to a commercial photodiode-array detector used in the fixed wavelength mode in terms of linearity, precision and detection limits. The chief advantages of the new device are small size, low power consumption, and low cost.     

单分子定位超分辨显微成像有机荧光探针的研究进展

在生物医学领域,对纳米尺寸级别的微小生物目标进行精确定位研究具有非常重要的意义,而光学显微成像技术为此提供了强有力的工具。 光学显微成像技术受到光学衍射极限的限制,难以分辨尺寸在衍射极限(<200 nm)以下的生物结构,无法直接获取微小生物结构信息,阻碍了生物医学的进一步发展。 近年来,随着纳米分辨显微成像技术的出现,新型荧光探针的开发、成像系统与设备的不断发展及成像算法不断完善地深入结合,促进了光学衍射极限以下尺寸微观目标的研究。 基于单分子定位的超分辨荧光显微成像(SMLM)包括光激活定位成像(PALM)与随机光学重构超分辨成像(STORM),将有机荧光探针与超分辨光学显微成像技术紧密结合在一起,荧光探针的光物理性质直接决定着超分辨成像结果的好坏。 因此,设计不同性能的荧光探针可以实现超精细结构的不同超分辨成像,为研究其生物学功能提供了有力的工具。 本文着重围绕基于SMLM的原理、有机荧光探针的设计要求、用于SMLM的荧光探针种类及其生物应用等方面进行总结综述,指出了单分子定位成像上存在的不足,并对其发展方向进行了展望,希望为对超分辨成像研究感兴趣或初涉该领域的研究者提供成像理论与探针设计方面的帮助。     

Fast spatially localised electrooptical mode in vertically aligned nematic LCs with negative dielectric anisotropy

ABSTRACT

Electro-optical switching and the liquid crystal (LC) director distribution are studied in spatially periodic electric field for vertically aligned LC with negative dielectric anisotropy. Two electro-optical switching modes characterised by different switching times are observed. These modes are well distinguished optically by choosing proper geometry for the polarisers axes orientation. One of these modes is significantly faster as compared to the other. The fast switching is explained in terms of localised near-to-surface director reorientation. The 3D-numerical simulation shows very good agreement with the experiment: it points out the existence of the disclination lines and field-stabilised walls responsible for the localised director field switching and its relaxation. Possibilities of enhancing the fast mode for high-speed light modulators are discussed.     

Comparison of the precision of peak areas in consecutive injections in the isocratic and gradient HPLC analysis of some pharmaceutical preparations

An isocratic and a gradient elution mode of HPLC are compared in the analysis of five multicomponent pharmaceutical formulations. It is shown that in some cases, these modes have no advantages over each other in the precision of the peak areas of components if sample solutions are injected successively. In other cases, the results of the gradient elution are more precise.     

Sulfoxide hemithioindigo tweezers – visible light addressable capture and release

Introducing responsive elements into supramolecular recognition systems offers great advantages for the control of intermolecular interactions and represents an important stepping stone towards multi-purpose and reprogrammable synthetic systems. Of particular interest is implementation of light-responsiveness because of the unique ease and precision of this signal. Here we present visible light responsive hemithioindigo-based molecular tweezers that bear a highly polar sulfoxide function as an additional recognition unit inside their binding site. Sulfur oxidation allows to simultaneously enhance all crucial properties of this receptor type i.e. photoswitching capability, thermal stability of individual switching states, binding affinity, and binding modulation upon switching. With a novel titration method the thermodynamic binding parameters were determined using reduced sample amounts. Employing these strongly enhanced molecular tweezers allowed to demonstrate photocontrol of intermolecular charge transfer in a reversible manner.

Hemithioindigo based molecular tweezers with a comprehensively improved property profile are obtained by simple oxidation of the sulfur atom.     

Spectral time moment analysis of microgel structure and dynamics

The structure and dynamics of crosslinked nanoparticles (microgels) made out of hydroxypropylcellulose (HPC) polymer chains were studied using dynamic light scattering spectroscopy. The microgel light scattering spectra were found to be highly nonexponential requiring a spectral time moment analysis in which the spectra were fit to a sum of stretched exponentials. Each term offers three parameters for analysis and represents a single spectral mode. At room temperature microgel spectra reveal three modes. Two faster modes are almost diffusive and correspond to apparent sizes of 25 and 450–650 nm. The slowest mode is independent of scattering angle and is reminiscent of the slow polymer mode observed in identical non‐crosslinked polymer solutions. When solution temperature is varied from 23 to 45°C and back, the microgel undergoes a reversible volume phase transition between 40 and 45°C. According to the time‐moment analysis, above the transition temperature two faster modes collapse into one with apparent hydrodynamic radius of 100–150 nm, while the slow mode remains largely unchanged. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 771–781, 2008     

In situ Activatable Peptide-based Nanoprobes for Tumor Imaging

Owing to its excellent biological properties, peptide has been widely used in the design of nanoprobes capable of enhancing tumor imaging signals. In recent years, a number of peptide-based nanoprobes with strong loading capacity and great biocompatibility have been developed for precision tumor imaging by coupling peptide motifs with different imaging agents. It is worth noting that, compared with "always on" mode, the use of stimulus-mediated in situ activatable mode to design and control the self-assembly or nanostructure transformation of peptide-based nanoprobes in vivo can achieve the significant improvement of imaging efficiency. Herein, we summarize the recent progress of in situ activatable peptide-based nanoprobes for tumor imaging in diverse imaging modes, including magnetic resonance imaging(MRI), fluorescence imaging(FI), photoacoustic imaging(PAI), radionuclide imaging(RI) and multimodal imaging. Finally, we briefly prospect the challenges and potential development directions of this field.     

Enhanced dSTORM imaging using fluorophores interacting with cucurbituril

Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes or proteins as fluorophores. The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy’s localization precision and imaging resolution. Fluorophores TAMRA and Atto Rho6G, which can interact with macrocyclic host cucurbit[7]uril (CB7) to form host-guest compounds, were found to improve the fluorescence intensity and lifetimes of these dyes. We enhanced the localization precision of direct stochastic optical reconstruction microscopy (dSTORM) by introducing CB7 into the imaging buffer, and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6G increase over 2 times.     

A new memory effect: smectic liquid crystalline memory having both conductive and optical memory

In order to compensate for light leakage in the oblique viewing directions of the dark state of the horizontal-switching mode such as the in-plane switching and fringe field switching modes. The viewing angle performance of the horizontal-switching mode with an compensation film has been evaluated on a case by case basis according to the retardation values of the films. Consequently, we found that a much better dark state can be achieved by using an optimised compensation film.     

Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain

The precision of single‐molecule localization‐based super‐resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV‐induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single‐color dSTORM. By screening 39 dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error‐free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15 nm was achieved. Caged SD‐dSTORM can resolve the ultrastructure of single 40 nm synaptic vesicles in brain sections similar to images obtained by immuno‐electron microscopy, yet with much improved label density in two independent channels.     

Extracting effective normal modes from equilibrium dynamics at finite temperature

A general method for obtaining effective normal modes of a molecular system from molecular dynamics simulations is presented. The method is based on a localization criterion for the Fourier transformed velocity time-correlation functions of the effective modes. For a given choice of the localization function used, the method becomes equivalent to the principal mode analysis (PMA) based on covariance matrix diagonalization. On the other hand, a proper choice of the localization function leads to a novel method with a strong analogy with the usual normal mode analysis of equilibrium structures, where the Hessian system at the minimum energy structure is replaced by the thermal averaged Hessian, although the Hessian itself is never actually calculated. This method does not introduce any extra numerical cost during the simulation and bears the same simplicity as PMA itself. It can thus be readily applied to ab initio molecular dynamics simulations. Three such examples are provided here. First we recover effective normal modes of an isolated formaldehyde molecule computed at 20 K in very good agreement with the results of a normal mode analysis performed at its equilibrium structure. We then illustrate the applicability of the method for liquid phase studies. The effective normal modes of a water molecule in liquid water and of a uracil molecule in aqueous solution can be extracted from ab initio molecular dynamics simulations of these two systems at 300 K.     

Simultaneous determination of fundamental parameters for ferroelectric liquid crystals

An analysis of an experimental technique which allows for a simultaneous determination of several parameters with special interest for ferroelectric liquid crystals is presented. These parameters are the spontaneous polarization, the rotational viscosity and the switching time. In addition, the susceptibility associated with the soft mode free of contributions related to the helicoidal structure can also be obtained. The experimental results for these parameters for the ferroelectric liquid crystal HDOBACEEC are reported. A comparison between the switching time values deduced from the rotational viscosity and those obtained by optical measurements is performed.     

Anharmonic C?H and C=O Interactions in Peptide and Sugar

C?H and C=O stretching modes are two among many structural and dynamic probes of proteins and peptides in condensed phases. Anharmonic properties of these two modes in peptide and sugar have been examined using a second-order perturbative vibrational approach. High order force constants were obtained and examined to ˉnd how crucial they are in determining the degree of mode localization and the nature of mode anharmonicity of the two stretching modes. It is found that the C?H mode is highly localized,and its diagonal anharmonicity is mainly determined by the mode itself. However, the C=O mode is largely delocalized, and the diagonal anharmonicity involves contributions from other modes. The o?-diagonal anharmonicity between C?D and C=O modes is found to be negative in deuterated species, di?ering from those of the non-deuterated ones. It is also found that inter-mode interaction between each of the two modes with low-frequency modes contribute signiˉcantly to the o?-diagonal anharmonicity. These low-frequency modes give rise to a network of energy relaxation or intramolecular vibrational energy redistribution pathways which can be used to examine temporal behavior of intramolecular vibration energy °ow, provided a femtosecond broadband two-dimensional infrared spectroscopy is available.     

Simultaneous measurements of the flow velocities in a microchannel by wide/evanescent field illuminations with particle/single molecules

A laser-induced fluorescence imaging method was developed to simultaneously measure flow velocities in the middle and near wall of a channel with particles or single molecules, by selectively switching from the wide field excitation mode to the evanescent wave excitation mode. Fluorescent microbeads with a diameter of 175 nm were used to calibrate the system, and the collisions of microbeads with channel walls were directly observed. The 175 nm microbeads velocities in the main flow and at 275 nm from the bottom of the channel were measured. The measured velocities of particles or single molecules in two positions in a microchannel were consistent with the calculated value based on Poiseuille flow theory when the diameter of a microbead was considered. The errors caused by Brownian diffusion in our measurement were negligible compared to the flow velocity. Single lambda DNA molecules were then used as a flowing tracer to measure the velocities. The velocity can be obtained at a distance of 309.0 +/- 82.6 nm away from bottom surface of the channel. The technique may be potentially useful for studying molecular transportation both in the center and at the bottom of the channel, and interactions between molecules and microchannel surfaces. It is especially important that the technique can be permitted to measure both velocities in the same experiment to eliminate possible experimental inconsistencies.     

Atomic force microscopy of microvillous cell surface dynamics at fixed and living alveolar type II cells

Scanning probe techniques enable direct imaging of morphology changes associated with cellular processes at life specimen. Here, glutaraldehyde-fixed and living alveolar type II (ATII) cells were investigated by atomic force microscopy (AFM), and the obtained topographical data were correlated with results obtained by scanning electron microscopy (SEM) and confocal microscopy (CM). We show that low-force contact mode AFM at glutaraldehyde-fixed cells provides complementary results to SEM and CM. Both AFM and SEM images reveal fine structures at the surface of fixed cells, which indicate microvilli protrusions. If ATII cells were treated with Ca²⁺ channel modulators known to induce massive endocytosis, changes of the cell surface topography became evident by the depletion of microvilli. Low force contact mode AFM imaging at fixed ATII cells revealed a significant reduction of the surface roughness for capsazepine and 2-aminoethoxydiphenyl-borate (CPZ/2-APB)-treated cells compared to untreated control cells (Rc of 99.7 ± 6.8 nm vs. Rc of 71.9 ± 4.6 nm for N = 22), which was confirmed via SEM studies. CM of microvilli marker protein Ezrin revealed a cytoplasmic localization of Ezrin in CPZ/2-APB-treated cells, whereas a submembranous Ezrin localization was observed in control cells. Furthermore, in situ AFM investigations at living ATII cells using low force contact mode imaging revealed an apparent decrease in cell height of 17% during stimulation experiments. We conclude that a dynamic reorganization of the microvillous cell surface occurs in ATII cells at conditions of stimulated endocytosis.     

Inorganic Nanomaterial for Biomedical Imaging of Brain Diseases

In the past few decades, brain diseases have taken a heavy toll on human health and social systems. Magnetic resonance imaging (MRI), photoacoustic imaging (PA), computed tomography (CT), and other imaging modes play important roles in disease prevention and treatment. However, the disadvantages of traditional imaging mode, such as long imaging time and large noise, limit the effective diagnosis of diseases, and reduce the precision treatment of diseases. The ever-growing applications of inorganic nanomaterials in biomedicine provide an exciting way to develop novel imaging systems. Moreover, these nanomaterials with special physicochemical characteristics can be modified by surface modification or combined with functional materials to improve targeting in different diseases of the brain to achieve accurate imaging of disease regions. This article reviews the potential applications of different types of inorganic nanomaterials in vivo imaging and in vitro detection of different brain disease models in recent years. In addition, the future trends, opportunities, and disadvantages of inorganic nanomaterials in the application of brain diseases are also discussed. Additionally, recommendations for improving the sensitivity and accuracy of inorganic nanomaterials in screening/diagnosis of brain diseases.