Quantitative DNA imaging in breast tumor cells by a Hadamard transform fluorescence imaging microscope.

This paper presents a novel Hadamard transform (HT) fluorescence imaging microscope by combining multiplexed imaging technique with a conventional upright fluorescence microscope for single-cell imaging and quantitative cellular analysis. The HT imaging microscope can provide 511 x 512-pixel single-cell image with high sensitivity within 21 s. In this study, the high potential value of the microscope in biomedical analysis has been demonstrated by using it to evaluate the malignancy degree of thirty cases of human breast tumors based on the measurements of cellular DNA contents, with conclusions highly accordant with pathological diagnosis. The results show that the HT microscope has the ability to analyze very small specimens and the capability of detecting very high ploidy cells, which are advantages over flow cytometry. The microscope was also successfully applied to cellular morphological analysis, and it was demonstrated that a significant linear relationship exists between tumor nuclear DNA contents and the nuclear area, and malignant and benign tumors are significantly different in both DNA contents and nuclear area. The reliability of the HT microscope in cellular DNA measurements was also investigated.     

高分辨阿达玛变换显微荧光图像分析(Ⅱ)单细胞DNA荧光图像生成、处理及自动定量分析研究

本研究把荧光显微镜，阿达玛变换多通道成像技术和计算机图象处理技术结合起来，建立了一种单细胞DNA荧光图像自动定量分析系统，对系统的成像原理、信号编码和解码方法及单细胞定量分析方法进行了讨论，分析结果表明，本系统提供的单细胞定量分析数据稳定可靠。     

阿达玛变换显微荧光成像在单细胞定量分析中的应用研究

本文探讨了用阿达玛变换（HT）显微荧光成像系统获得基于256灰度级图像的单细胞荧光强度和解码定标值之间的关系,将归一化的HT图像用于定量分析,建立了适合不同样品的定量分析方法.在此条件下,定量分析数据有很好的准确度和精密度;将系统用于单个乳腺肿瘤细胞的DNA定量分析,对乳腺肿瘤的良恶性及癌变程度进行判断,分析结果与病理学诊断结论一致.     

Hadamard transform fluorescence image microscopy using one-dimensional movable mask

With a 511-slit one-dimensional (1D) Hadamard mask and a highly sensitive linear charge-coupled device (CCD), spatial multiplexing is performed and a programmable Hadamard transform (HT) microscopic fluorescence imaging system was developed. The system can generate 511×512 pixel format images for small samples. Sensitivity, signal to noise ratio, imaging speed and spatial resolution of this system were discussed. The results show that the system can be applied for single-cell imaging sensitively in a short time. Spatial resolution up to 0.24 μm/pixel, which is close to the resolution limit of the conventional optical microscope, has been obtained under oil lens. The weak native fluorescence imaging for pollen cells can be realized within 1 min. The system has been applied for multi-parameter evaluation of tumor malignancy based on nuclear DNA ploidy measurements for one breast tumor specimen. The result indicates that the system has good application prospect in cell biology and medicine.     

微流控进样阿达玛变换显微荧光成像单细胞分析

将微流控芯片用于单细胞进样,以自制阿达玛变换显微荧光成像分析系统进行成像检测,讨论了影响单细胞进样和荧光成像的主要因素,并对花粉细胞DNA含量进行定量分析。结果表明：微流控进样技术与HT显微成像技术相结合,可有效可靠地应用于单细胞分析中,所测定的三个玉帘花粉的DNA含量分别为124．4、123．9和62．9pg。     

高分辨阿达玛变换显微荧光图像分析(Ⅲ)——系统分辨率和图像恢复过程对单细胞分析结果的影响

提出了一种改进的阿达玛变换(HT)显微荧光图像分析系统,以单细胞试样分析为基础,分别对系统的分辨率和解码后的图像恢复过程进行了讨论.结果表明,该系统可应用于单细胞形态分析和定量分析.图像在x和y方向的像素分辨率相同,并达到了同一成像物镜下的空间分辨率水平,因此在获取微米级单细胞试样的微弱荧光信号的二维图像时,系统的成像能力较好,可用于单细胞形态分析.对花粉细胞的荧光衰退过程的定量分析结果表明,对不同HT图像提供的同一系列试样的定量数据进行比较时,必须对所有该系列试样的图像恢复过程进行归一化处理.     

使用一维机械模板的高分辨阿达玛变换显微图像探测技术研究

阿达玛变换（HT）是一种类似于傅里叶变换（FT）的光谱调制技术,具有多通道同时检测和多通道成像能力等优点,但两者的数学模型、对光信号的调制方法和调制手段都不一样。由于HT仅涉及四则运算,而FT涉及较为复杂的三角函数和复数运算,所以HT的解码速度快于FT。在成像技术方面,HT具有直接成像的能力,而FT只能对通过其它方式获取的图像进行加工处理。     

阿达玛变换显微图象分析(Ⅱ)──荧光衰减对图象和定量分析数据的影响

采用计算机模拟的方法,系统地考察了试样的荧光衰减对阿达玛变换图象和定量分析数据的影响,并把阿达玛变换显微图象分析仪用于细胞内DNA的定量分析,得到了满意的结果。     

显微荧光－阿达玛变换三维图象分析

把阿达玛变换多通道成象技术与荧光显微镜结合起来,首次成功地获得了细胞的阿达玛变换三维荧光图象,该图象能同时提供微小物体中某一物质的含量及分布等信息。     

阿达玛变换显微图象分析系统测定乳腺肿瘤细胞DNA含量

本文以吖啶橙为细胞DNA荧光探针, 探讨了影响阿棕玛显微图象分析仪定量分析细胞DNA时的细胞荧光强度以及影响人乳腺肿瘤细胞DNA定量分析结果准确性的几个因素, 结果表明: (1)乙醇是理想的固定剂, 醛类固定剂对细胞AO-DNA复合物的荧光有显著影响; (2)吖啶橙染色液中含Triton X-100时细胞荧光强芳明显增大; (3)吖啶橙的最佳荧光染色浓度为50μg/mL; (4)在分析乳腺肿瘤细胞DNA含量(倍性)时, 采用外标法(以人血涂片或淋巴结细胞涂上的淋巴细胞为标准二倍体细胞)或内标法(以乳腺肿瘤细胞涂片上的正常上皮细胞为标准二倍体细胞)均能得到较好的结果, 但后者更为可靠; (5)随机分析六十个以上肿瘤细胞, 可得到较好分析结果。     

Instrument for Hadamard transform three-dimensional fluorescence microscope image analysis

An instrument combining fluorescence microscopy with Hadamard transform multiplexed imaging was designed by which a three-dimensional Hadamard transform fluorescence microscopic cell image was obtained. The image can provide useful information including, simultaneously, the apparent dimensions and the shape of the analytical sample, the content and the distribution of some species in it.     

阿达玛变换显微图象分析系统在乳腺肿瘤细胞DNA倍性分析中的应用研究

以正常人外周静脉血淋巴细胞为标准二倍体细胞，以吖啶橙为细胞DNA荧光探针，用阿达玛变换显微图象分析仪测定了六例乳腺肿瘤的细胞DNA含量（倍性），分析结果与病例学诊断结论吻合，表明该仪器可望用于乳腺癌的诊断和预后研究．研究结果还表明：乳腺肿瘤细胞DNA倍性与核面积（象素）呈显著正相关．     

In situ spectral imaging of marker proteins in gastric cancer with near-infrared and visible quantum dots probes

This study presents the investigation of bioconjugating ability of near-infrared (NIR) CdSeTe/ZnS quantum dots (QDs) (710 nm) and visible CdSe QDs (595 nm) in immunofluorescent staining for cancer biomarkers in gastric cancer tissues probed with the homemade Hadamard transform (HT) spectral imaging microscope and a commercial multispectral imaging system. The results show that imunostaining ability of NIR QDs probes is stronger than that of visible QDs when the two kinds of QDs are simultaneously used to probe the cancer biomarkers such as cytokeratin 20 (CK20) and proliferating cell nuclear antigen (PCNA) in gastric cancer tissues. Moreover, when the two QDs probes are used for immunostaining successively for the same target molecules, staining order has great influences on the final results due to their different conjugating ability to the marker proteins. The results imply that NIR QDs hold more promise for real-time imaging of tumor tissues due to its higher sensitivity and contrast. In addition, the results also demonstrate the potential of Hadamard transform spectral imaging as a useful tool in biomedical analysis and quantitative evaluation for tumor tissues.     

高分辨阿达玛变换显微荧光图像分析(Ⅰ)——系统的成像能力及其在单个肿瘤细胞分析中的应用

阿达玛变换(Hadamard transform, HT)是一种类似于傅里叶变换的光谱调制技术, 具有多通道同时检测和多通道成像能力. 实现高分辨HT成像的关键在于阿达玛模板的制作, 阿达玛模板有两种, 即移动式机械编码模板(Movable mechanical mask)和固定式光电模板(Stationary electro-optic mask). 在实际成像方面, 移动模板和固定模板各有优缺点: 前者一般用石英玻璃制作, 对光信号不会因模板吸收而导致信号损失, 因此数据很可靠, 而且模板的制作也较为容易, 但由于采用步进电机驱动而容易导致机械故障, 难以实现快速编码; 后者无移动部件, 无机械故障, 因此系统比较紧凑, 但由于它是由液晶材料制成的(可导致信号损失), 从而限制了其在某些光谱区域的使用. 此外, 它对系统的软件设计要求比前者高, 实现高分辨成像更加困难. 正是由于上述原因, 实现快速、高分辨HT成像具有一定难度, 最近有关HT成像技术的报道极少.     

阿达玛变换光谱和成像技术的应用和研究进展

阿达玛变换(HT)作为一种多通道光谱调制技术，具有多通道同时检测能力、多通道成像能力以及适用于数据处理等优点。综述了近十年来HT光谱和成像技术在分析科学中的应用和研究进展。主要从HT模板编码技术和HT激发序列应用技术等方面讨论了其最新发展和存在的问题，并展望了其发展前景。     

Hadamard变换显微图象分析III. 单细胞内DNA,RNA和蛋白质的 分析

本文用自制的Hadamard变换显微图象分析仪分析了AO和FITC染色的单细胞的荧光光谱,用该仪器获取了单个洋葱细胞核内DNA的分布图象,并在535nm处分别测定了AQ染色的鸡红血细胞及洋葱表皮细胞核的荧光强度。所得结果与生物学结果吻合,显示了该仪器在单细胞试样分析中的应用价值。     

Study on Schiff base complexes-cellular DNA interactions by a novel system of Hadamard transform fluorescence image microscopy

A novel system of Hadamard transform microscopic fluorescence imaging for single cells is presented, based on which the DNA ploidy of rat hepatocyte was quantitatively measured. The result shows that diploid rat hepatocyte has a stable DNA content, thus diploid rat hepatocyte was used to investigate the binding of five clinical anticancer agents, vincristine, cyclophosphamide, nitrogen mustard, cis-diamminedichloroplatinum(n) (CDDP) and mitomycin-C, with cellular DNA when acridine orange (AO) was used as the competitive fluorescence probe. Based on this model, some Schiff base complexes-cellular DNA interactions were investigated. The results indicate that all the twenty-two compounds, including Schiff base ligands of N-2-hydroxy-naphthaldehyde with D-glucoamine (NG) and the complexes of 3d-transitional metals ions with NO and with D-glucoamine (Glu) and the mixed complexes of NG and Glu series with alpha-glycine (GNG), have the ability to enter the cell membrane and interact with cellular DNA. Four of the compounds, CuGlu, Fe(II)NG, Fe(III)NG and CuGluG can intercalate with DNA like AO does and depress AO-DNA fluorescence to 70% or lower. An in intro UV-visible spectroscopic study on the compound-DNA spectra testified the above results and suggests that diverse interaction mechanisms coexist for all these complexes except intercalating mode. This study presents a new in vitro method for initial screening of anticancer compounds.     

Preparation of liposomes loaded with quantum dots, fluorescence resonance energy transfer studies, and near-infrared in-vivo imaging of mouse tissue

We report on a simple, fast and convenient method to engineer lipid vesicles loaded with quantum dots (QDs) by incorporating QDs into a vesicle-type of lipid bilayer using a phase transfer reagent. Hydrophilic CdTe QDs and near-infrared (NIR) QDs of type CdHgTe were incorporated into liposomes by transferring the QDs from an aqueous solution into chloroform by addition of a surfactant. The QD-loaded liposomes display bright fluorescence, and the incorporation of the QDs into the lipid bilayer leads to enhanced storage stability and reduced sensitivity to UV irradiation. The liposomes containing the QD were applied to label living cells and to image mouse tissue in-vivo using a confocal laser scanning microscope, while NIR images of mouse tissue were acquired with an NIR fluorescence imaging system. We also report on the fluorescence resonance energy transfer (FRET) that occurs between the CdTe QDs (the donor) and the CdHgTe QDs (the acceptor), both contained in liposomes. Based on these data, this NIR FRET system shows promise as a tool that may be used to study the release of drug-loaded liposomes and their in vivo distribution.

Detecting and Removing Data Artifacts in Hadamard Transform Ion Mobility-Mass Spectrometry Measurements

Applying Hadamard transform multiplexing to ion mobility separations (IMS) can significantly improve the signal-to-noise ratio and throughput for IMS coupled mass spectrometry (MS) measurements by increasing the ion utilization efficiency. However, it has been determined that fluctuations in ion intensity as well as spatial shifts in the multiplexed data lower the signal-to-noise ratios and appear as noise in downstream processing of the data. To address this problem, we have developed a novel algorithm that discovers and eliminates data artifacts. The algorithm employs an analytical approach to identify and remove artifacts from the data, decreasing the likelihood of false identifications in subsequent data processing. Following application of the algorithm, IMS-MS measurement sensitivity is greatly increased and artifacts that previously limited the utility of applying the Hadamard transform to IMS are avoided. Figure

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吖啶橙-细胞DNA荧光抑制法筛选抗癌药物

在DNA荧光法‘’‘初步筛选抗癌药物的实验中,漠化乙锭（EB）是常用的荧光探针,其本身荧光板弱,但能与双链DNA发生专一性插入作用,使荧光大幅度地增强．但EB不能进入完整的细胞膜［’j．对陕橙（AO）常用于细胞内DNA和RNA的定量测定,并能用于活细胞染色;AO与核酸的结合方式有2种：一种是在嵌入核酸双链的碱基对之间形成AO－DNA复合物,另一种是与单链核酸的磷酸发生静电间相互作用,形成AO－RNA复合物．如用蓝光激发,前者发射峰波长为530urn的绿色荧光,后者发射峰波长为640urn的红色荧卅’‘．本文提出一种对细胞周期非…