离焦系统下的单分子三维取向研究

提出了一种新思路来确定单分子的三维取向,在全内反射荧光显微成像的基础上,在显微镜的油镜上加一个水层,水和油的折射率不同造成光程差,产生离焦,利用全内反射产生隐失波激发了单分子的纵向分量,并在探测器前增加了一个分光镜,把光路分成偏振方向相互垂直的两束光,使得探测器上的光场分布包含单分子三维取向的信息。推导了隐失场激发的偶极矩光场在探测器上的光强分布表达式,通过分析偏振方向相互垂直的两束光在探测器上的不同的强度分布,来判断单分子偶极矩的三维取向。

超分辨远场生物荧光成像——突破光学衍射极限

长期以来,远场光学荧光显微镜凭借其非接触、无损伤、可探测样品内部等优点,一直是生命科学中最常用的观测工具。但由于衍射极限的存在,使传统的宽场光学显微镜横向和纵向的分辨率分别仅约为230 nm和1000 nm。为了揭示细胞内分子尺度的动态和结构特征,提高光学显微镜分辨率成为生命科学发展的迫切要求,在远场荧光显微镜的基础上,科学家们已经发展出许多实用的提高分辨率甚至超越分辨率极限的成像技术。例如,采用横向结构光照明提高横向分辨率到约100 nm,利用纵向驻波干涉效应将纵向分辨率提高5~10倍。然而,直到在光学荧光显微镜中引入非线性效应后,衍射极限才被真正突破,如受激荧光损耗显微镜利用非线性效应实现了30~50 nm的三维分辨率。另外应用荧光分子之间能量转移共振原理以及单荧光分子定位技术也可以突破衍射极限,甚至可以将分子定位精度提高到几个纳米的量级。

Morphological and ultrastructural changes in tobacco BY-2 cells exposed to microcystin-RR

Tobacco BY-2 cells were exposed to microcystin-RR (MC-RR) at two concentrations, 60 mu g mL(-1) and 120 mu g mL(-1), to study the changes in morphology and ultrastructure of cells as a result of the exposure. Exposure to the lower concentration for 5 d led to typical apoptotic morphological changes including condensation of nuclear chromatin, creation of a characteristic 'half moon' structure, and cytoplasm shrinkage and decreased cell volume, as revealed through light microscopy. fluorescence microscopy, and transmission electron microscopy, respectively. Exposure to the higher concentration, on the other hand, led to morphological and ultrastructural changes typical of necrosis, such as rupture of the plasma membrane and the nuclear membrane and a marked swelling of cells. The presence of many vacuoles containing unusual deposits points to the involvement of vacuoles in detoxifying MC-RR. Results of the present study indicate that exposure of tobacco BY-2 cells to MC-RR at a lower concentration (60 mu g mL(-1)) results in apoptosis and that to a higher concentration (120 mu g mL(-1)), in necrosis. (C) 2009 Elsevier Ltd. All rights reserved.

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity. (C) 2007 Elsevier Inc. All rights reserved.

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells. (C) 2004 Published by Elsevier B.V.

High PL-efficiency ZnO nanocrystallites/PPV composite nanofibers

In this paper, we report for the first time on the synthesis of ZnO nanocrystallites in conjugated polymer (PPV) nanofibers by the coupling of the in situ/blend methods and electrospinning. These composite nanofibers were characterized by fluorescence microscopy, atomic force microscope (AFM), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), photoluminescence (PL) spectra, Fourier transform infrared (FT-IR) spectroscopy, and X-ray powder diffraction (XRD).

Core Crosslinking of Biodegradable Block Copolymer Micelles Based on Poly(ester carbonate)

A biodegradable amphiphilic block copolymer, PEG-b-P(LA-co-MAC), was used to prepare spherical micelles consisting of a hydrophobic P(LA-co-MAC) core and a hydrophilic PEG shell. To improve their stability, the micelles were crosslinked by radical polymerization of the double bonds in the hydrophobic blocks. The crosslinked micelles had similar sizes and a narrow size distribution compared to their uncrosslinked precursor. The improved stability of the crosslinked micelles was confirmed by measurements of the CMC and a thermodynamic investigation. These micelles can internalize into Hela cells in vitro as demonstrated by inverted fluorescence microscopy and CLSM. These stabilized nanoscale micelles have potential use in biomedical applications such as drug delivery and disease diagnosis.

Rapid self-assembly of oligo(o-phenylenediamine) into one-dimensional structures through a facile reprecipitation route

The self-assembly of oligo(o-phenylenediamine) (OPD) into 1-D nanostructures on a macroscopic length scale was found when they were transferred from N-methyl pyrrolidone to deionized water. Field emission scanning electron microscopy and confocal fluorescence microscopy were used to investigate the morphology of the precipitates. Results showed that large amounts of OPD 1-D supertructures could be obtained through the simple reprecipitation route, and the length of the fibers could be tuned from microscale to macroscale by adjusting the ratio of two solvents. X-ray diffraction patterns and UV-vis spectra revealed that pi-pi interactions between OPD molecules that facilitated the formation of 1-D structures became predominant when they were transferred from a good solvent to a bad one. Accordingly, a possible formation mechanism was proposed.

Conformation change of horseradish peroxidase in lipid membrane

The electrochemical behavior of horseradish peroxidase (HRP) in the dimyristoyl phosphatidylcholine (DMPC) bilayer on the glassy carbon (GC) electrode was studied by cyclic voltammetry. The direct electron transfer of HRP was observed in the DMPC bilayer. Only a small cathodic peak was observed for HRP on the bare GC electrode. The electron transfer of HRP in the DMPC membrane is facilitated by DMPC membrane. UV-Vis and circular dichroism (CD) spectroscopy were used to study the interaction between HRP and DMPC membrane. On binding to the DMPC membrane the secondary structure of HRP remains unchanged while there is a substantial change in the conformation of the heme active site. Tapping mode atomic force microscopy (AFM) was first applied for the investigation on the structure of HRP adsorbed on supported phospholipid bilayer on the mica and on the bare mica. HRP molecules adsorb and aggregate on the mica without DMPC bilayer. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed in the DMPC bilayer. The adsorption of HRP in the DMPC bilayer changes drastically the domains and defects in the DMPC bilayer due to a strong interaction between HRP and DMPC films.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

Detection of constitutive homomeric associations of the integrins Mac-1 subunits by fluorescence resonance energy transfer in living cells

Integrins alpha(M)beta(2) plays important role on leukocytes, such as adhesion, migration, phagocytosis, and apoptosis. It was hypothesized that homomeric associations of integrin subunits provide a driving force for integrins activation, and simultaneously inducing the formation of integrins clusters. However, experimental reports on homomeric associations between integrin subunits are still controversial. Here, we proved the homomeric associations of the isolated Mac-1 subunits in living cells using three-channel fluorescence resonance energy transfer (FRET) microscopy and FRET spectra methods. We found that the extent of homomeric associations between beta(2) subunits is higher than alpha(M) subunits. Furthermore, FRET imaging indicated that the extent of homomeric associations of the Mac-1 subunits is higher along the plasma membrane than in the cytoplasm. Finally, we suggested that homomeric associations of the transmernbrane domains or/and cytoplasmic domains may provide the driving force for the formation of constitutive homomeric associations between alpha(M) or beta(2) subunits. (c) 2006 Elsevier Inc. All rights reserved.

Temporal characteristic simulation for stimulated emission depletion microscopy

The imaging technology of stimulated emission depletion (STED) utilizes the nonlinearity relationship between the fluorescence saturation and the excited state stimulated depletion. It implements three-dimensional (3D) imaging and breaks the diffraction barrier of far-field light microscopy by restricting fluorescent molecules at a sub-diffraction spot. In order to improve the resolution which attained by this technology, the computer simulation on temporal behavior of population probabilities of the sample was made in this paper, and the optimized parameters such as intensity, duration and delay time of the STED pulse were given.