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(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。     

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。     

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。     

Analysis of DNA in Single Pollen Cell by Hadamard Transform Microscopic Fluorescence Imaging

A noncommercial Hadamard transform microscopic image analysis system was used to measure fluorescence emitted by single cells. A method for subtracting the native fluorescence of theZephyranthes candida(Lindl.) Herb. pollen cell was developed based on its correlation to pollen area. Acridine orange and ethidium bromide were used as fluorescence probes for pollen DNA quantitative analysis, and the results obtained by the two methods were compared.     

阿达玛变换光谱和图象及其在分析化学中的应用

本文对阿达玛变换光谱和成象的原理进行了简单介绍，并对８０年代以来阿达玛变换技术在分析化学中的应用进行了评述。     

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.     

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…     

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．     

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.     

Studies on Hadamard Transform Fluorimetry

Hadamard transform spectroscopy has been investigated since the later 1960's and early 1970's. It was demonstrated that the application of Hadamard transform technique to spectroscopy can facilitate multichannel detection of weak signal. It can be appplied not only to component analysis, but to image analysis. Now, it has been widely used in infrared spectrometry, Raman spectrometry and photothermal deflection analysis. But its application to molecular fluorimetry has not been reported up to now.     

Multielement atomic absorption analysis using Hadamard transform spectroscopy with a new computation and superposition procedure

The application of Hadamard transform spectroscopy (HTS) to analytical atomic spectroscopy has been investigated. A theoretical examination of the signal to noise ratio indicates that HTS offers a slight multiplex advantage over single slit scanning of the spectrum for the measurement of intense lines, as in atomic absorption, but is disadvantageous for measuring small signals in atomic emission and fluorescence. Using a simple HT spectrometer Mg and Pb were determined simultaneously by flame atomic absorption spectrometry. Sensitivities similar to those of conventional systems were obtained but, owing to instrumental imperfections and a short data collection time, detection limits were worse. Optimum system performance was obtained when the mask interval width was equal to the width of the image of the spectrometer entrance slit. Greater spectral detail was revealed by superposition of a set of computed spectra in which the starting point of each spectrum was displaced from the others by a distance less than the interval width of the Hadamard mask. This approach gave improved spectra without increased instrumental complexity.     

双编码阿达玛变换光谱法：原理,仪器及光谱分析研究

本文根据阿达玛变换的原理研制成一种双编码阿达玛变换光谱仪，与单编码及常规光谱仪相比，该仪器具有高信噪比、高输入和单检测器多通道同时检测的优点。试样分析表明：该仪器能用于荧光谱分析和原子线光谱分析。     

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.     

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。     

High-resolution Hadamard transform microscope fluorescence imaging: quantifying the DNA content in single cells

This study presents a novel miniaturized Hadamard transform fluorescence imaging microscope, by combining a conventional fluorescence microscope with Hadamard transform multiplexing encoding using a one-dimensional movable mask to realize spatial resolution and a linear CCD for multichannel detection. The microscope can provide high-resolution automatically-generated 0–255 gray level HT images for morphological analysis and visualization of a single cell, and normalized HT images for cellular quantitative measurements. The microscopes imaging capability was applied to measure the DNA content in human lymphocyte, chicken erythrocyte and eel erythrocyte, and a comparative study was performed. The results show that the calibrated DNA content in a chicken erythrocyte is 2.32 pg when human lymphocyte is used as the standard, and eel erythrocyte may be a potentially reliable and novel standard for determining DNA contents in other species because it has a stable DNA value of 2.06 pg, with a CV of 4.3% when 20 eel erythrocytes are measured. The results also demonstrate that the HT imaging microscope should be valuable in the fields of medicine and cell biology.     

Hadamard transform capillary electrophoresis combined with laser-induced fluorometry using electrokinetic injection

Hadamard transform capillary electrophoresis (HTCE) based on electrokinetic injection allows laser-induced fluorescence detection using a small laser, namely the laser-diode-pumped YAG laser, as an excitation source. A small hole is fabricated at the center of a capillary by laser ablation; this hole functions as an inlet port for a sample solution. Therefore, the sample solution can be introduced electrophoretically into the capillary through the small hole. Multiple sample injection is accomplished by introducing a buffer solution from the end of the capillary and the sample solution through the hole. Both solutions are injected using two sets of high-voltage power supplies and migrate toward the opposite end of the capillary. A fluorescent analyte, rhodamine B, is successfully detected in the case of both single and multiple injection according to the Hadamard sequence code. By transforming the data encoded by the Hadamard matrix, the decoded data showed an increase in the signal-to-noise (S/N) ratio by a factor of 9.8. In the case of the sample containing two amino acids labeled with rhodamine B isothiocyanate (RBITC), although the concentration of every component including free RBITC is lower than the concentration limit of detection obtained by single injection, a substantial improvement in the sensitivity is achieved and all components are identified by the Hadamard transform technique.     

Detection of 27 molecules in a single injection volume by Hadamard transform capillary electrophoresis.

A concentration detection limit of 100 fM was achieved for the fluorescein ion by improving the experimental setup used for Hadamard transform capillary electrophoresis. Two argon-ion lasers, a gating laser for sample injection and a probe laser for the excitation of analyte molecules, were employed for the efficient photodegradation of analyte molecules in laser-induced fluorescence detection using an optically gated sample-injection method. In addition, a dichroic mirror, located in the pathway of the probe laser was used to exclude the other lines of the argon-ion laser. Using a Hadamard matrix on the order of 2046, the concentration limit of detection for fluorescein ion was determined to be 100 fM at S/N = 3, in which the average number of molecules in a single injection volume was calculated to be 27. The influences of the output power in both the gating and probe lasers on the sensitivity are also discussed.     

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.