超短光脉冲的平均功率和脉宽及锁模状态对双光子显微镜成象的影响

显微镜成象是研究生物活体细胞结构及活动规律的重要技术.飞秒光脉冲是多光子显微镜的主要光源,它的性能(脉宽、平均功率和锁模状态等)严重影响成象的质量.双光子激发荧光强度正比于超短脉冲平均功率的平方,反比于脉宽,锁模稳定状态严重影响成象的清晰度.对用Cameleon转染的HeLa细胞和GFP转基因水稻叶的实验研究结果与理论一致.     

Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals - Quantum Dots (QDs) - have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties - wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.     

New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye

Background

To date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems.

Results

Here we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology.

Conclusions

NeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.     

Simple expressions for performance parameters of complex filters, with applications to super-Gaussian phase filters

To study the three-dimensional (3-D) behavior produced by complex filters, we have extended the expressions for the axial and the transverse gain to the case in which the best image plane is not near the paraxial focus. Super-Gaussian phase filters are proposed to control the 3-D image response of an optical system. Super-Gaussian phase filters depend on several parameters that modify the shape of the phase filter, producing tunable control of the 3-D response of the optical system. The filters are capable of producing a wide range of optical effects: transverse superresolution with high depth of focus, 3-D superresolution, and transverse apodization with different axial responses.     

Photothermal image flow cytometry in vivo

The capability of photothermal (PT) microscopy to image moving, unlabeled cells in real time in vivo is demonstrated in a study of circulating red and white blood cells in blood and lymph microvessels of rat mesentery. Potential applications of this optical tool, called PT flow cytometry, are discussed.     

Tuneable edge enhancement filters for optical correlation

Difference of Gaussian (DOG) filters, a form of wavelet filter, are an extremely useful preprocessing tool for the enhancement of image edge data. The DOG filter is highly tuneable by control of the standard deviations of its constituent Gaussian distributions. These can be used in the frequency plane of optical correlators and implemented in the form of a static non-updateable filter. This article reports on a simple optical technique using photorefractive materials whereby a filter, updateable in real time and very similar to the DOG filter, is implemented by tuning the intensity of the hologram reference wave to give enhanced modulation in a selectable frequency band. This is called a tuneable photorefractive (TPR) filter. The results of the DOG and TPR filters are compared with those of the phase-only filter and classical matched spatial filter with respect to the criteria of signal-to-noise ratio, Horner efficiency and discrimination capability.     

Real-time recognition of microscopic phase objects

A method of image enhancement and real-time input of 3-D, microscopic phase objects into a coherent optical pattern recognition system is described. The method consists of directing a low-power laser beam into a microscope objective to produce a real, magnified, coherent image of the specimen under test. The image plane is followed by two successive Fourier transform (FT) planes. In the first FT plane, low and high frequency spatial filters, one of which is photographically produced, are used as pre-processing filters to enhance the image quality. The enhanced signal is imaged from the first FT plane to the second FT plane which contains a matched spatial filter used for specimen identification. The system does not require an expensive incoherent-to-coherent light transducer and in addition, is capable of utilizing both phase and amplitude information from 3-D objects. Examples of results are given.     

Development of a Spin-Exchange Optical Pumping-Based Polarized ~3He System at the China Spallation Neutron Source(CSNS)

Polarized ~3He neutron spin filters(NSFs) can be used as a vital tool for neutron polarization production and analysis.The China Spallation Neutron Source(CSNS),as one of the major neutron facilities in China,has committed resources to the development of a polarized ~3He NSF program to support its growing polarized neutron research.A spin-exchange optical pumping(SEOP)-based polarized ~3He system and other necessary hardware for NSF transport has been recently developed.The performance of the system is benchmarked using an in-house developed cell named "Trident".Neutron beam measurements yield a ~3 He polarization of 77% with over 200 h of on-beam relaxation time.Combining this newly developed SEOP system with the recently reported cell fabrication station,CSNS is now capable of the fully self-sustained production of ~3He NSFs that shall support its future neutron polarization research.     

光学空间滤波过程的计算机仿真

提出了一种利用MATLAB软件并通过计算机仿真光学空间滤波实验过程的新方法.其特点是:既可以随意改变所设计滤波器的参量,又可以对输入图象进行振幅、相位或复合滤波,并且可实现傅里叶变换频谱中相位信息的提取、存储和利用,因而能够完成一般光学实验中往往难以实现的某些操作.并分别给出了网格滤波、低通、高通及相位滤波等仿真实验结果.这种仿真实验给光学滤波器的设计和图象处理带来很大方便,同时也为相关器件的设计提供了一条新的途径.     

Green Fluorescent Protein (GFP) as a Marker for Cell Viability After UV Irradiation

Generation of Chinese Hamster Ovary (CHO) cell lines stably expressing green fluorescent protein (GFP) was achieved using a plasmid vector that encoded the red-shifted pCX-xGFP under the control of a strong hybrid promoter composed of a CMV enhancer and a -actin/-globin gene promoter. Cotransfection of the promoter-less pSV2-Neo helper plasmid transmitting neomycin resistance was followed by selection with the antibiotic G418. Constitutive GFP expression could be visualized in living and fixed cells using fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. DNA repair-proficient (AA8) and deficient (UV5) CHO strains were used for survival tests after UVC irradiation. Cells carrying the GFP construct (AA8-pGFP, UV5-pGFP) show the same response to UV irradiation (colony forming ability) as their nontransformed parental cell lines (AA8, UV5). Using GFP as a marker for cell viability, cells were harvested after certain postirradiation growth periods and the numbers of GFP expressing cells and fluorescence intensities were determined by FACS analysis. Generally, GFP fluorescence in irradiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuously (leading to a dose-dependent increase in GFP contents).     

基于PC的光电探测器设计平台

提出了一种光电探测器设计平台,它基于VB工具,在PC机基础上整合了光电探测器光谱灵敏度测试系统与滤色片组匹配设计系统,具有对光电池光谱灵敏度、有色玻璃的光谱透过率、滤色片组与光电池构成的光电探测器的光谱灵敏度等参数的测试功能,可对光电探测器滤色片(组)进行设计,提供了光电探测器设计与测试的一体化平台。     

光电混合相关识别系统中匹配滤波器缩放比例的确定方法

在光电混合匹配滤波相关识别系统中,由于输入图像的频谱与匹配滤波器的形成方式不同,不能直接实现对应频率成份的准确对准。这个问题需要通过对匹配滤波器进行缩放来解决。根据透镜的傅里叶变换性质和计算全息理论,分别推导出输入图像的频谱的表达式和滤波空间光调制器加载了匹配滤波器后的透射函数的表达式,并根据这两个表达式得到匹配滤波器的缩放比例的公式,然后在实际的光学识别系统中对根据该公式计算的缩放比例进行了验证。实验结果表明,在该缩放比例下,相关峰的质量有较大改善,这说明此时输入图像的频谱与匹配滤波器对应频率成份实现了较好地对准。     

Monitoring of mechanical properties of serially passaged bovine articular chondrocytes by atomic force microscopy

Atomic force microscopy (AFM) is a powerful tool in imaging cells and tissues and probing their mechanical properties. Articular chondrocytes, the cells responsible for the production and maintenance of cartilaginous extracellular matrix in the knee joint, change their morphology and dedifferentiate during in vitro expansion culture. It was unclear if the mechanical properties of chondrocytes change accompanying phenotype variation. The elasticity of in vitro serially cultured bovine articular chondrocytes was investigated using AFM. The chondrocytes changed their morphology from round to spindle-like. The freeze-dried P0 chondrocytes showed significantly higher modulus than did the serially passaged (P1–P4) chondrocytes. The change of chondrocyte morphology was accompanied with a decrease of elastic modulus.     

Analysis of cell surface molecular distributions and cellular signaling by flow cytometry

Flow cytometry is a fast analysis and separation method for large cell populations, based on collection and processing of optical signals gained on a cell-by-cell basis. These optical signals are scattered light and fluorescence. Owing to its unique potential ofStatistical data analysis and sensitive monitoring of (micro)heterogeneities in large cell populations, flow cytometry—in combination with microscopic imaging techniques—is a powerful tool to study molecular details of cellular signal transduction processes as well. The method also has a widespread clinical application, mostly in analysis of lymphocyte subpopulations for diagnostic (or research) purposes in diseases related to the immune system. A special application of flow cytometry is the mapping of molecular interactions (proximity relationships between membrane proteins) at the cell surface, on a cell-by-cell basis. We developed two approaches to study such questions; both are based ondistance-dependent quenching of excited state fluorophores (donors) by fluorescent or dark (nitroxide radical) acceptors via Förstertype dipole-dipole resonance energy transfer (FRET) and long-range electron transfer (LRET) mechanisms, respectively. A critical evaluation of these methods using donor- or acceptor-conjugated monoclonal antibodies (or their Fab fragments) to select the appropriate cell surface receptor or antigen will be presented in comparison with other approaches for similar purposes. The applicability of FRET and LRET for two-dimensional antigen mapping as well as for detection of conformational changes in extracellular domains of membrane-bound proteins is discussed and illustrated by examples of several lymphoma cell lines. Another special application area of flow cytometry is the analysis of different aspects of cellular signal transduction, e.g., changes of intracellular ion (Ca²⁺, H⁺, Na⁺) concentrations, regulation of ion channel activities, or more complex physiological responses of cell to external stimuli via correlated fluorescence and scatter signal analysis, on a cell-by-cell basis. This way different signaling events such as changes in membrane permeability, membrane potential, cell size and shape, ion distribution, cell density, chromatin structure, etc., can be easily and quickly monitored over large cell populations with the advantage of revealing microheterogeneities in the cellular responses. Flow cytometry also offers the possibility to follow the kinetics of slow (minute- and hour-scale) biological processes in cell populations. These applications are illustrated by the example of complex flow cytometric analysis of signaling in extracellular ATP-triggered apoptosis (programmed cell death) of murine thymic lymphocytes.     

One-mode model for patterned metal layers inside integrated color pixels

Optimized design of the optical filters inside integrated color pixels (ICPs) for complementary metal-oxide semiconductor image sensors requires analytical models. ICP optical filters consist of subwavelength patterned metal layers. We show that a one-mode model, in which subwavelength gaps in the metal layer are described in terms of single-mode waveguides, suffices to predict the salient features of measured ICP wavelength selectivity. The Airy-like transmittance formula, derived for transverse-electric polarization, predicts an angle-independent cutoff wavelength, which is in good agreement with predictions made with a two-dimensional finite-difference time-domain method.     

Scanning Near-Field Optical Microscopy and Microspectroscopy of Green Fluorescent Protein in Intact Escherichia coli Bacteria

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical information and constitutes an important new technique for visualizing biological systems. By coupling a spectrograph to a near-field microscope, we have been able to perform microspectroscopic measurements with a spatial resolution greatly exceeding that of the conventional optical microscope. Here we present SNOM images of Escherichia coli bacteria expressing a mutant green fluorescent protein (GFP), an important reporter molecule in cell, developmental, and molecular biology. Near-field emission spectra confirm that the fluorescence detected by SNOM arises from bacterially expressed GFP molecules.     

彩色图像的非相干光相关识别

提出一种用非相干光系统识别彩色图像的方法,将脸色图像分解成三个单色像处理,设计一可实现的仅相位光学传递函数,对其进行带能优化,人而提高信噪比并使相关峰锐化,通过一对滤波器来合成该光学传递函数,获得了采色图像相关识别的实验结果。     

成像过程的计算机模拟

按照阿贝二次成像理论,物体成像经历了两个过程：（１）物面上光场复振幅的空间分布,变换成光学系统后焦面上的频率分布;（２）后焦面上的频率分布还原成像面上的光场复振幅空间分布。本文介绍了如何用计算模拟这两个过程,设计的空间滤波器可以随意改变频谱分布,并可观察到像面上对应的变化。     

GFP immunogold staining, from light to electron microscopy, in mammalian cells

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one hand it allows adequate fixation for EM analysis maintaining GFP antigenicity, on the other hand it also enables the epoxy resins inclusion after immunogold staining. Both of them help to preserve better the ultrastructure. However GFP immunogold staining presents some drawbacks, such as the progressive decrease in immunogold labeling with tissue depth. Special attention must be taken when using GFP-tagged protein, since the fusion could interfere with their localization and function. In this review we provide a detailed protocol of the GFP immunogold staining, their main applications for EM and possible troubles.     

A novel approach to quantitative ultrasonic naked gene delivery and its non-invasive assessment

The purpose of this study was to investigate practical, safe, easy-to-use, non-cytotoxic, and reliable parameters to apply to an ultrasound (US) naked gene therapy system. The ultrasound pressure at the point of cell exposure was measured using a calibrated hydrophone and the intensity calculated. An acoustic power meter calibrated using a hydrophone was used to measure the power of the transducer. Four cell types were exposed to US with different exposure times and intensities. Fluorescent microscopy, spectrophotometry, scanning electron microscope, laser scanning confocal microscopy, flow cytometry and histogram analysis were used to evaluate the results of the study. The plasmid of green fluorescent protein (GFP) served as the reporter gene. The energy accumulation E in US gene delivery for 90% cell survival was defined as the optimal parameters (E=3.56+/-0.06), and at 80% cell survival was defined as the damage threshold (E=59.67+/-3.54). US safely delivered GFP into S180 cells (35.1 kHz) at these optimal parameters without obvious damage or cytotoxity in vitro. Exposed cell function was proved normal in vivo. The transfection rate was 35.83+/-2.53% (n=6) in viable cells, corresponding to 90.17+/-1.47% (n=6) cell viability. The intensity of GFP expression showed a higher fluorescent peak in the group of adeno-associated virus GFP vector (AVV-GFP) than in the control group (P<0.001). The effect of US gene delivery and cell viability correlated as a fifth order polynomial with US intensity and exposure time. With optimal parameters, US can safely deliver naked a gene into a cell without damage to cell function. Both optimal uptake and expression of gene depend on the energy E at 90% cell survival. E can be applied as a control factor for bioeffects when combined with other parameters. Stable caviation results in optimal parameters for gene delivery and the transient caviation may cause cell damage, which will bring about a sharp rise of permeabilization. The results may be applied to the development of a novel clinical gene therapeutic system.