阿达玛变换显微图象分析Ⅰ──细胞定量分析初探

简单介绍了阿达玛变换多通道成象技术的原理及我们研制的阿达玛变换显微图象分析仪的工作原理,在此基础上,讨论了该仪器用于细胞定量分析及图象分析的可行性,包括图象和分析结果的可靠性及外界条件时分析结果的影响。结果表明：阿达玛变换显微图象分析是一种灵敏、准确、受外界干扰小、能同时提供分析物图象和定量分析结果的有效的细胞定量分析方法。     

阿达玛变换显微图象分析Ⅰ──细胞定量分析初探

简单介绍了阿达玛变换多通道成象技术的原理及我们研制的阿达玛变换显微图象分析仪的工作原理,在此基础上,讨论了该仪器用于细胞定量分析及图象分析的可行性,包括图象和分析结果的可靠性及外界条件对分析结果的影响。结果表明：阿达玛变换显微图象分析是一种灵敏、准确、受外界干扰小、能同时提供分析物图象和定量分析结果的有效的细胞定量分析方法。     

对同一细胞内DNA、RNA和蛋白含量相关测定的显微荧光光度法

用荧光染料DAPI、PyroninY和FITC分别染同一细胞内DNA、RNA和蛋白.在紫外光、绿光和兰光顺序激发后,用MPVⅡ显微荧光光度计测量反映单个细胞内DNA、RNA和蛋白含量的荧光强度.根据荧光发射光谱分析,每种染料荧光之间的干扰是可以忽略的.测量结果与FCM获得的结果是一致的.显微荧光光度术对单个细胞多参数相关测量的优点是简单、便宜,并可用于对活细胞获得形态学和定量细胞化学的组合信息.     

彗星系统定量检测柠檬醛损伤黄曲霉DNA的研究

理化因素致细胞DNA损伤,彗星测试提供了一个直观的方法。采用新型SCGE图象分析系统(IMI10),将细胞显微分光光度分析与显微成像及图象分析结合,直接检测柠檬醛致黄曲霉核DNA损伤,与国际流行的SCGE图象分析系统相比,具有分析速度快、便于分析,同时具有中英文可切换界面和多格式输出打印特点。该系统使彗星试验的检测时间缩短2/3,并提高了准确性,可实现对活细胞多种结构参数、细胞内分子与膜的变化状况同时进行长时间连续的动态瞬间监测,具有广阔应用前景。     

梨实生树不同发育区的叶片细胞学研究

采用显微分光光度计和显微图像分析仪比较研究了8年生梨实生树(Pyrus pyrifliaNakai)童区和成年区叶片细胞核 DNA含量、RNA含量和细胞、细胞核面积大小的差异。梨实生树从童区向成年区转变后,叶片内细胞核DNA含量上升,细胞内RNA合成加强,细胞和细胞核面积增大;同时,叶肉组织结构分化程度提高,叶面积增大,叶片加厚。     

应用图象分析仪及显微分光光度计分析转化细胞的一些生物学特性

本文用扫描显微分光光度计(西德,Opton)观察比较了两例经紫外线和化学诱变剂(ENU或DMBA)转化的人胚胃、肾细胞系的形态特征,用测微荧光法原位定量细胞核的DNA,用仪器的图象分析系统测量细胞和细胞核的周长、面积、最大直径、核质比等几何参数,测定结果对转化细胞及正常细胞生物学特性的鉴定,提供了某些独特的定量指标,取得了良好的效果。     

适用于细胞荧光测量的多道显微荧光计

建立了由倒置荧光显微镜和光学多道分析仪(OMA)连接而组成的适用于细胞荧光测量的多道显微荧光计,编制了数据处理程序。利用这一装置测量了单个细胞,多细胞的荧光光谱和拓扑(topography)。和传统的显微荧光计相比,该装置具有测量灵敏度和精度高、速度快等特点,可用来进行活细胞动态过程的研究。     

平阳霉素(争光霉素A_5)对艾氏腹水癌细胞核酸代谢及其作用机制之探讨

艾氏腹水癌小鼠腹腔内注入平阳霉素1/6半致死剂量,腹水癌细胞DNA合成受到抑制。随注入药物时间之延长,DNA合成呈直线下降。但在同一剂量和时间条件下,对腹水癌细胞RNA合成率没有影响。通过放射自显影和MPV II显微分光光度计,测定了S期细胞百分数和单个S期细胞中~3H-TdR放射自显影银粒,说明DNA合成率之抑制不是由于S期细胞百分数之降低,而是由于单个S期细胞中DNA合成强度之下降。进一步用MPV II显微分光光度计对同一S期细胞中放射自显影银粒和孚尔根萤光定量测定,发现平阳霉素能引起早S期细胞积累;同时早S期细胞DNA合成抑制也较为明显。说明早S期细胞对平阳霉素较为敏感。其机制可能与早S期细胞合成之DNA富含GC组分有关。这与博莱霉素优先和富含GC组分的多聚核苷酸结合的结果是一致的。     

平阳霉素(争光霉素A_5)对艾氏腹水癌细胞核酸代谢及其作用机制之探讨

艾氏腹水癌小鼠腹腔内注入平阳霉素1/6半致死剂量,腹水癌细胞DNA合成受到抑制。随注入药物时间之延长,DNA合成呈直线下降。但在同一剂量和时间条件下,对腹水癌细胞RNA合成率没有影响。通过放射自显影和MPV Ⅱ显微分光光度计,测定了S期细胞百分数和单个S期细胞中~3H-TdR放射自显影银粒,说明DNA合成率之抑制不是由于S期细胞百分数之降低,而是由于单个S期细胞中DNA合成强度之下降。进一步用MPV Ⅱ显微分光光度计对同一S期细胞中放射自显影银粒和孚尔根萤光定量测定,发现平阳霉素能引起早S期细胞积累;同时早S期细胞DNA合成抑制也较为明显。说明早S期细胞对平阳霉素较为敏感。其机制可能与早S期细胞合成之DNA富含GC组分有关。这与博莱霉素优先和富含GC组分的多聚核苷酸结合的结果是一致的。     

显微图象分析仪DNA定量分析中涂片与印片效果比较

显微图象分析仪ＤＮＡ定量分析中涂片与印片效果比较朱建伟田玉旺＊张庆慧＊＊刘爱茹冉玫（山东省中医药研究所病理科,济南２５００１４＊北京军区总院病理科＊＊山东医科大学病理教研室）用显微图像分析仪进行瘤细胞ＤＮＡ定量分析时,染色效果,背景清晰度,对照细胞的...     

Distinct modulated pupil function system for real-time imaging of living cells

Optical microscopy is one of the most contributive tools for cell biology in the past decades. Many microscopic techniques with various functions have been developed to date, i.e., phase contrast microscopy, differential interference contrast (DIC) microscopy, confocal microscopy, two photon microscopy, superresolution microscopy, etc. However, person who is in charge of an experiment has to select one of the several microscopic techniques to achieve an experimental goal, which makes the biological assay time-consuming and expensive. To solve this problem, we have developed a microscopic system with various functions in one instrument based on the optical Fourier transformation with a lens system for detection while focusing on applicability and user-friendliness for biology. The present instrument can arbitrarily modulate the pupil function with a micro mirror array on the Fourier plane of the optical pathway for detection. We named the present instrument DiMPS (Distinct optical Modulated Pupil function System). The DiMPS is compatible with conventional fluorescent probes and illumination equipment, and gives us a Fourier-filtered image, a pseudo-relief image, and a deep focus depth. Furthermore, DiMPS achieved a resolution enhancement (pseudo-superresolution) of 110 nm through the subtraction of two images whose pupil functions are independently modulated. In maximum, the spatial and temporal resolution was improved to 120 nm and 2 ms, respectively. Since the DiMPS is based on relay optics, it can be easily combined with another microscopic instrument such as confocal microscope, and provides a method for multi-color pseudo-superresolution. Thus, the DiMPS shows great promise as a flexible optical microscopy technique in biological research fields.     

Modification and optimization of PCR methods for detection of MIE region from human herpesvirus 5

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.     

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences.     

一个新的脑电信号分析系统：小波分析理论的运用

小波变换是一种把时间、频率（或尺度）两域结合起来的分析方法。它被誉为“分析信号的数学显微镜”。本系统将小波变换用于脑电信号分析,是一个在Ｗｉｎｄｏｗｓ３．１下开发的脑电分析系统。     

Instruments for rapid cytophotometric analysis of morphologically identified clinical cell material

Two instruments for cytophotometric analysis are described. The first is a generally applicable object stage scanning microspectrophotometer for the wavelength region 240-700 nm. The microscopic image is shown on a monitor screen and delineation of the analyzed objects is made with the aid of a light pen. The second instrument is a high speed microscope photometer specially adapted for the measurement of DNA, nuclear protein, and projected nuclear area following combined Feulgen and Naphthol Yellow S staining. A two-dimensional CCD sensor measures the light intensity in the microscopic image with a resolution of 0.3 micron. The objects are selected by the operator pointing the light pen at the image screen. The integrated absorbance at two different wavelengths, together with the projected area, is automatically measured for each object and presented in histogram form. Cells in clusters or sections can be measured under operator control with the help of the light pen. The time required for measuring 100 cells is 5-10 min in an ordinary preparation. The application of these instruments to the grading of cellular atypia and in the prognostic evaluation of malignant tumors is demonstrated on material from developing squamous bronchial carcinoma and in invasive mammary and prostatic adenocarcinoma.     

Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry

From a variety of analytical electron microscopy experiments, the chromosomes of dinoflagellates are known to contain sizeable amounts of cations, the latter thought to contribute to the neutralization of the negative charge carried by the phosphate groups in the DNA backbone. From previous Ca and Mg chelation experiments, it is also known that these cations are necessary for the compaction and preservation of the chromosome architecture. Similar conclusions have been recently presented by our group concerning mammalian mitotic chromosomes, in studies based on secondary ion mass spectrometry (SIMS) carried out with the University of Chicago high-resolution scanning ion microprobe (UC-SIM). We have now applied this instrument to image the distribution of DNA-bound Ca(2+) and Mg(2+) in dinoflagellate chromosomes, a goal that could not be attained earlier by analytical electron microscopy. Analyzed quantitatively and imaged here by SIMS for the first time, through their cation content, are the chromosomes of the dinoflagellates Prorocentrum micans, Gymnodinium mikimotoi and Gymnodinium dorsum. The cell nuclei were isolated and prepared for SIMS analysis with a minimal protocol (mechanical fractionation in culture medium followed by ethanol drying), which did not expose the samples to artifact-creating, alien chemical agents. By this approach, we have confirmed the earlier findings by several authors, and contributed new structural information provided by our ion probe capability to erode the sample surface layer by layer (SIMS tomography). Dinoflagellates, due to the absence of histones, represent an ideal model system where cations may bind directly with DNA, allowing comparisons to be made with recently reported X-ray crystallography results at atomic resolution. Such comparisons yielded quantitative confirmation that the Ca(2+)+Mg(2+) concentrations found for e.g. P. micans are consistent with those anticipated to provide complete charge neutralization of naked DNA by cations, also resulting in maximal DNA compaction.     

Plant Genome Size Measurement with DNA Image Cytometry

To test the reliability of DNA image cytometry for the measurement of nuclear DNA content in plant material, we conducted independent experiments in two laboratories using different image analysis instruments for densitometric measurement of nuclear DNA amount in Feulgen-stained squash preparations of root tips. The 2C nuclear DNA content of the nine species studied spanned a 100-fold range (approx. 0.3–33 pg). The estimates of nuclear DNA content measured with image cytometry methods were comparable to values obtained previously using both photometric cytometry and flow cytometry. Image cytometry methods showed little variation among repeated experiments within each laboratory or among different operators using the same instrument. Furthermore, the interphase-peak method (measurement of several hundred interphase nuclei per slide) was comparable to the classical prophase/telophase approach (measurement of ten early prophase and ten late telophase nuclei per slide). Hence, DNA image cytometry gives accurate and reproducible results and may be used as an alternative to photometric cytometry in plant nuclear DNA content measurements. In the present study, we propose that two standards for quality control of nuclear DNA content measurement are used in plant DNA image cytometry: (1) the coefficient of variation of the peak should be lower than 6%, and (2) the 4C/2C ratio should be between 1.9 and 2.1.     

A scanning device for the double beam leitz interference microscope

Summary The Leitz double beam interference microscope and its microscope photometer attachment have been rebuilt to a scanning instrument. The wide separation between the reference and object beam enables measurements of optical path differences (opd) in relatively large microscopic objects or in closely packed smaller objects without difficulties caused by the lack of free space for the reference beam. This advantage can now be combined with that of a scanning device. Using the system developed integrated values of the opd and hence the dry mass content of heterogeneous microscopic objects can be determined.Scanning in interference contrast is carried out in the image plane with a 0.1 mm diaphragm driven by two direct current motors. The intensity of the light passing the diaphragm is kept constant by adjustment of the glass wedge in the reference beam by means of a servo system. The displacement of the wedge, being proportional to the opd between the microscopic object and the free background, is recorded and integrated. The final value given by the integrator is proportional to the dry mass of the microscopic object.     

Use of Comet assay to assess DNA damage in patients infected by Helicobacter pylori: comparisons between visual and image analyses

Studies of DNA damage in gastric epithelial cells of Helicobacter pylori (H. pylori)-infected patients are conflicting, possibly due to different methods used for scoring DNA damage by Comet assay. Therefore, we compared the sensitivity of visual microscopic analysis (arbitrary units-scores and comets%) and image analysis system (tail moment), in the gastric epithelial cells from the antrum and corpus of 122 H. pylori-infected and 32 non-infected patients. The feasibility of cryopreserved peripheral blood lymphocytes and whole-blood cells for DNA damage biomonitoring was also investigated. In the antrum, the levels of DNA damage were significantly higher in H. pylori-infected patients with gastritis than in non-infected patients with normal mucosa, when evaluated by image analysis system, arbitrary units and comets%. In the corpus, the comets% was not sufficiently sensitive to detect the difference between H. pylori-infected patients with gastritis and non-infected patients with normal mucosa. The image analysis system was sensitive enough to detect differences between non-infected patients and H. pylori-infected patients with mild gastritis and between infected patients with moderate and severe gastritis, in both antrum and corpus, while arbitrary units and comets% were unable to detect these differences. In cryopreserved peripheral blood lymphocytes, the levels of DNA damage (tail moment) were significantly higher in H. pylori-infected patients with moderate and severe gastritis than in non-infected patients. Overall, our results indicate that the image analysis system is more sensitive and adequate to measure the levels of DNA damage in gastric epithelial cells than the other methods assayed.     

Emerging technologies in mass spectrometry imaging

Mass spectrometry imaging (MSI) as an analytical tool for bio-molecular and bio-medical research targets accurate compound localization and identification. In terms of dedicated instrumentation, this translates into the demand for more detail in the image dimension (spatial resolution) and in the spectral dimension (mass resolution and accuracy), preferably combined in one instrument. At the same time, large area biological tissue samples require fast acquisition schemes, instrument automation and a robust data infrastructure. This review discusses the analytical capabilities of an "ideal" MSI instrument for bio-molecular and bio-medical molecular imaging. The analytical attributes of such an ideal system are contrasted with technological and methodological challenges in MSI. In particular, innovative instrumentation for high spatial resolution imaging in combination with high sample throughput is discussed. Detector technology that targets various shortcomings of conventional imaging detector systems is highlighted. The benefits of accurate mass analysis, high mass resolving power, additional separation strategies and multimodal three-dimensional data reconstruction algorithms are discussed to provide the reader with an insight in the current technological advances and the potential of MSI for bio-medical research.