Influence Of Micro-Impurity On Protein Crystal Growth Studied By The Etch Figure Method

The investigation of the effect of micro impurity on crystal growth by optical microscopy has been validated. The results showed that the growth rate of a lysozyme crystal was affected even if the concentration of impurity of fluorescent-labeled lysozyme (abbreviation, F-lysozyme) was very small. Different concentrations of F-lysozyme had different effects on crystal growth rate. The growth rate decreased much more as F-lysozyme concentration increased. The density of incorporated F-lysozyme on different grown layers of a lysozyme crystal during crystal growth was obtained from the results of flat-bottomed etch pits density. (C) 2008 Elsevier B.V. All rights reserved.

Direct observation of adsorption sites of protein impurities and their effects on step advancement of protein crystals

We measured noninvasively step velocities of elementary two-dimensional (2D) islands on {110} faces of tetragonal lysozyme crystals, under various supersaturations, by laser confocal microscopy combined with differential interference contrast microscopy. We studied the correlation between the effects of protein impurities on the growth of elementary steps and their adsorption sites on a crystal surface, using three kinds of proteins: fluorescent-labeled lysozyme (F-lysozyme), covalently bonded dimers of lysozyme (dimer), and a 18 kDa polypeptide (18 kDa). These three protein impurities suppressed the advancement of the steps. However, they exhibited different supersaturation dependencies of the suppression of the step velocities. To clarify the cause of this difference, we observed in situ the adsorption sites of individual molecules of F-lysozyme and fluorescent-labeled dimer (F-dimer) on the crystal surface by single-molecule visualization. We found that F-lysozyme adsorbed preferentially on steps (i.e., kinks), whereas F-dimer adsorbed randomly on terraces. Taking into account the different adsorption sites of F-lysozyme and F-dimer, we could successfully explain the different effects of the impurities on the step velocities. These observations strongly suggest that 18 kDa also adsorbs randomly on terraces. Seikagaku lysozyme exhibited a complex effect that could not alone be explained by the two major impurities (dimer and 18 kDa) present in Seikagaku lysozyme, indicating that trace amounts of other impurities significantly affect the step advancement.

Constitutive plasma membrane targeting and microdomain localization of Dok5 studied by single-molecule microscopy

In the present study, single-molecule fluorescence microscopy was used to examine the characteristics of plasma membrane targeting and microdomain localization of enhanced yellow fluorescent protein (eYFP)-tagged wild-type Dok5 and its variants in living Chinese hamster ovary (CHO) cells. We found that Dok5 can target constitutively to the plasma membrane, and the PH domain is essential for this process. Furthermore, single-molecule trajectories analysis revealed that Dok5 can constitutively partition into microdomain on the plasma membrane. Finally, the potential mechanism of microdomain localization of Dok5 was discussed. This study provided insights into the characteristics of plasma membrane targeting and microdomain localization of Dok5 in living CHO cells. (C) 2008 Elsevier B.V. All rights reserved.

Heterodimerization of integrin Mac-1 subunits studied by single-molecule imaging

Heterodimerization of integrin Mac-1 (alpha(M) beta(2)) Subunits plays important role on regulating leukocytes adhesion to extracellular matrix or endothelial cells. Here, using total internal reflection microscopy, we investigated the heterodimerization of integrin Mac-1 subunits at the single-molecule level in live cells. Individual alpha(M) subunit fused to the enhanced yellow fluorescent protein (eYFP) was imaged at the basal plasma membrane of live Chinese hamster ovary (CHO) cells. Through analysis of mean square displacement (MSD), diffusion coefficient, the size of restricted domain and fraction of molecules undergoing restricted diffusion, we found that as compared with the diffusion in the absence of beta(2) subunit, the diffusion of single-molecule of alpha(M)-YFP was suppressed significantly in the presence of beta(2) subunit. Thus, based on the oligomerization-induced trapping model, we suggested that in the presence of beta(2) subunit, the am subunit may form heterodimer with it. (C) 2008 Elsevier Inc. All rights reserved.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (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.

Optical properties of transparent alkali gallium silicate glass-ceramics containing Ni2+-doped beta-Ga2O3 nanocrystals

Broadband near-infrared (IR) luminescence in transparent alkali gallium silicate glass-ceramics containing N2+-doped beta-Ga2O3 nanocrystals was observed. This broadband emission could be attributed to the T-3(2g) (F-3) -> (3)A(2g) (F-3) transition of octahedral Ni2+ ions in glass-ceramics. The full width at half-maximum (FWHM) of the near-IR luminescence and fluorescent lifetime of the glass-ceramic doped with 0.10 mol% NiO were 260 nm and similar to 1220 mu s, respectively. It is expected that transparent Ni2+-doped beta-Ga2O3 glass-ceramics with this broad near-IR emission and long fluorescent lifetime have potential applications as super-broadband optical amplification media.

K9玻璃薄膜对离子交换磷酸盐玻璃波导表面保护的优化研究

采用镀K9玻璃薄膜方法来解决离子交换掺铒磷酸盐玻璃波导表面的侵蚀问题,对K9玻璃薄膜的厚度进行了优化研究。测量分析了样品的荧光光谱和荧光寿命,采用光学显微镜和棱镜耦合技术对不同K9玻璃薄膜厚度下制备波导的表面形貌和导光特性进行了表征和测试。结果表明,与掺铒磷酸盐玻璃原材料相比,镀K9玻璃薄膜后荧光光谱保持不变,荧光寿命稍有下降(约0.2 ms);K9玻璃薄膜的厚度在60~80 nm的范围内保护效果最佳。为下一步制备掺铒有源玻璃光波导器件奠定了良好的实验基础。

傅立叶分解法荧光层析成像

本文提出一种基于结构光照明和傅立叶分解方法的荧光层析成像技术，该技术首先将激发光的强度沿着光轴方向调制成余弦函数，然后用此激发光对样品作传统的二维扫描，在每一个扫描位置余弦函数的频率在一定的范围内扫描，同时一一对应地记录下所发出的荧光强度。只要对所纪录的荧光序列做一个简单的傅立叶变换，即可以得到此位置样品沿着光轴方向的荧光团分布。这样通过一个传统的二维扫描，就可以得到一个三维的阳样品分布。

掺铒碲酸盐玻璃的热力学稳定性和光谱性质的研究

分析了掺Er^3+碲酸盐玻璃的热力学稳定性能，研究了掺Er^3+碲酸盐玻璃的吸收和荧光光谱性质；应用Judd-Ofelt理论计算了碲酸盐玻璃中Er^3+离子的强度参数Ω(Ω2=4．79×10^-20cm^2,Ω4=1．52×10^-20cm^2，Ω6=0．66×10^-20cm^2)，计算了离子的自发跃迁几率，荧光分支比；应用McCumber理论计算了Er^3+的受激发射截面(σe=10．40×10^-21cm^2)、Er^3+离子^4I13／2→^4I15／2发射谱的荧光半高宽(FWHM=65．5nm)

一种新型掺铒碲酸盐玻璃的光谱性质研究

研究了一种新型掺Er^3+碲酸盐玻璃的光谱性质；应用Judd-Ofelt理论计算了碲酸盐玻璃中Er^3+离子的强度参数Ω(Ω2=4．79×10^-20cm^2，Ω4=1．52×10^-20cm^2，Ω6=0．66×10^-20cm^2)，计算了离子的自发跃迁概率，荧光分支比；应用McCumber理论计算了Er^3+的受激发射截面(σe=10．40×10^-21cm^2)，Er^3+离子^4I13/2→^4I15/2发射谱的荧光半高宽(FWHM=65．5nm)及各能级的荧光寿命(^4I13/2能级为τrad

Intense infrared luminescence in transparent glass-ceramics containing beta-Ga2O3 : Ni2+ nanocrystals

Transparent glass-ceramics containing beta-Ga2O3:Ni2+ nanocrystals were synthesized and characterized by X-ray diffraction, transmission electron microscopy, and electron energy loss spectroscopy. Intense broad-band luminescence centering at 1200 nm was observed when the sample was excited by a diode laser at 980 nm. The room-temperature fluorescent lifetime was 665 mu s, which is longer than the Ni2+-doped ZnAl2O4 and LiGa5O8 glass-ceramics and is also comparable to the Ni2+-doped LiGa5O8 single crystal. The intense infrared luminescence with long fluorescent lifetime may be ascribed to the high crystal field hold by Ni2+ and the moderate lattice phonon energy of beta-Ga2O3. The excellent optical properties of this novel material indicate that it might be a promising candidate for broad-band amplifiers and room-temperature tunable lasers.