高光谱成像技术在生物医学中的应用

高光谱成像技术是传统成像与光谱技术相结合的一门新技术,其可同时获得被测物体的空间特征与光谱信息,以实现对物质特性的研究。本文介绍了高光谱成像技术的基本原理、系统的基本构成及特点,总结和阐述了近年来高光谱成像技术在生物医学领域的发展,以及其在疾病诊断中的应用。     

Oil Adulteration Identification by Hyperspectral Imaging Using QHM and ICA

To investigate the feasibility of identification of qualified and adulterated oil product using hyperspectral imaging(HIS) technique, a novel feature set based on quantized histogram matrix (QHM) and feature selection method using improved kernel independent component analysis (iKICA) is proposed for HSI. We use UV and Halogen excitations in this study. Region of interest(ROI) of hyperspectral images of 256 oil samples from four varieties are obtained within the spectral region of 400–720nm. Radiation indexes extracted from each ROI are used as feature vectors. These indexes are individual band radiation index (RI), difference of consecutive spectral band radiation index (DRI), ratio of consecutive spectral band radiation index (RRI) and normalized DRI (NDRI). Another set of features called quantized histogram matrix (QHM) are extracted by applying quantization on the image histogram from these features. Based on these feature sets, improved kernel independent component analysis (iKICA) is used to select significant features. For comparison, algorithms such as plus L reduce R (plusLrR), Fisher, multidimensional scaling (MDS), independent component analysis (ICA), and principle component analysis (PCA) are also used to select the most significant wavelengths or features. Support vector machine (SVM) is used as the classifier. Experimental results show that the proposed methods are able to obtain robust and better classification performance with fewer number of spectral bands and simplify the design of computer vision systems.     

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged¹. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact². We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use³. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm^-1) is lower than that of water (72 dynes cm^-1) and also below the limit (25 - 30 dyne cm^-1) for stomatal penetration⁴, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO₂ and O₂, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation^5,6, surgery⁷, artificial blood⁸, oxygenation of growth media⁹, and studies of ice crystal formation in plants¹⁰. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.     

不同产地和不同采收期荷叶中生物碱的含量变化

本文建立高效液相色谱法同时测定荷叶中三种阿朴啡类生物碱2-羟基-1-甲氧基阿朴啡、N-去甲荷叶碱及荷叶碱的含量,并比较了6个产地(湖南、湖北、浙江、陕西、贵州、北京)和5个采收期(北京6～10月)荷叶中这三种生物碱的含量。结果显示不同产地荷叶生物碱含量差别明显,但均以荷叶碱为主,三者的总量在0.11%～1.15%之间,其中以浙江、贵州和湖南产荷叶中的三种生物碱含量较高,而湖北、陕西和北京产荷叶中生物碱含量较低。不同采收期对荷叶生物碱的含量也有较大影响,6月荷叶生物碱总量最高,随生长时间的延长,荷叶碱和N-去甲荷叶碱的含量降低,而2-羟基-1-甲氧基阿朴啡含量增高。     

Rapid Particle Size Measurement Using 3D Surface Imaging

The present study introduces a new three-dimensional (3D) surface image analysis technique in which white light illumination from different incident angles is used to create 3D surfaces with a photometric approach. The three-dimensional features of the surface images created are then used in the characterization of particle size distributions of granules. This surface image analysis method is compared to sieve analysis and a particle sizing method based on spatial filtering technique with nearly 30 granule batches. The aim is also to evaluate the technique in flowability screening of granular materials. Overall, the new 3D imaging approach allows a rapid analysis of large amounts of sample and gives valuable visual information on the granule surfaces in terms of surface roughness and particle shape.     

鲜叶保存方法对茶树基因组DNA提取效果的影响

探讨一种简便有效的鲜叶保存方法,对克服茶树DNA研究中远距离采样的困难具有重要意义．为此,以槠叶齐品种的鲜梢为材料,分别采用-20℃、-70℃、硅胶脱水干燥3种方法保存样品,采用改进的CTAB法与SDS法分别对3种方法保存的样品及对照样(鲜叶)进行基因组DNA的提取与纯化,对所得DNA的质量进行多重检测的结果表明,硅胶脱水干燥法保存的样品与其他几种方法保存的样品一样,都提取获得了高质量的DNA,因此,硅胶脱水干燥法可作为远距离采样制备茶树DNA的一种较好的鲜叶保存方法,具有操作简单、经济实用、不受时空等条件限制的特点,该方法同样适用于其他植物．     

Growth Simulation and Discrimination of Botrytis cinerea,Rhizopus stolonifer and Colletotrichum acutatum Using Hyperspectral Reflectance Imaging

This research aimed to develop a rapid and nondestructive method to model the growth and discrimination of spoilage fungi, like Botrytis cinerea, Rhizopus stolonifer and Colletotrichum acutatum, based on hyperspectral imaging system (HIS). A hyperspectral imaging system was used to measure the spectral response of fungi inoculated on potato dextrose agar plates and stored at 28°C and 85% RH. The fungi were analyzed every 12 h over two days during growth, and optimal simulation models were built based on HIS parameters. The results showed that the coefficients of determination (R²) of simulation models for testing datasets were 0.7223 to 0.9914, and the sum square error (SSE) and root mean square error (RMSE) were in a range of 2.03–53.40×10⁻⁴ and 0.011–0.756, respectively. The correlation coefficients between the HIS parameters and colony forming units of fungi were high from 0.887 to 0.957. In addition, fungi species was discriminated by partial least squares discrimination analysis (PLSDA), with the classification accuracy of 97.5% for the test dataset at 36 h. The application of this method in real food has been addressed through the analysis of Botrytis cinerea, Rhizopus stolonifer and Colletotrichum acutatum inoculated in peaches, demonstrating that the HIS technique was effective for simulation of fungal infection in real food. This paper supplied a new technique and useful information for further study into modeling the growth of fungi and detecting fruit spoilage caused by fungi based on HIS.     

Detection and Discrimination of Cotton Foreign Matter Using Push-Broom Based Hyperspectral Imaging: System Design and Capability

Cotton quality, a major factor determining both cotton profitability and marketability, is affected by not only the overall quantity of but also the type of the foreign matter. Although current commercial instruments can measure the overall amount of the foreign matter, no instrument can differentiate various types of foreign matter. The goal of this study was to develop a hyperspectral imaging system to discriminate major types of foreign matter in cotton lint. A push-broom based hyperspectral imaging system with a custom-built multi-thread software was developed to acquire hyperspectral images of cotton fiber with 15 types of foreign matter commonly found in the U.S. cotton lint. A total of 450 (30 replicates for each foreign matter) foreign matter samples were cut into 1 by 1 cm2 pieces and imaged on the lint surface using reflectance mode in the spectral range from 400-1000 nm. The mean spectra of the foreign matter and lint were extracted from the user-defined region-of-interests in the hyperspectral images. The principal component analysis was performed on the mean spectra to reduce the feature dimension from the original 256 bands to the top 3 principal components. The score plots of the 3 principal components were used to examine clusterization patterns for classifying the foreign matter. These patterns were further validated by statistical tests. The experimental results showed that the mean spectra of all 15 types of cotton foreign matter were different from that of the lint. Nine types of cotton foreign matter formed distinct clusters in the score plots. Additionally, all of them were significantly different from each other at the significance level of 0.05 except brown leaf and bract. The developed hyperspectral imaging system is effective to detect and classify cotton foreign matter on the lint surface and has the potential to be implemented in commercial cotton classing offices.     

高光谱成像技术对人体面部和手掌的成像及光谱分析

目的：应用高光谱成像技术对正常人体不同部位图像进行采集,分析这些部位的光谱特征,得到正常值数据,并为该技术用于疾病诊断和中医面诊和手诊奠定基础。方法：使用高光谱成像仪,采集10例（男5女5）健康人的面部和双手掌图像,应用高光谱处理分析软件,对面和手掌划分15个分析区域,统计各分区从450-900 nm每间隔10 nm一个光谱段的各个分析区光强度值和光谱特征,并分析个体特点。结果：在面和手掌的高光谱图像上可以清晰地显示面部器官和手掌的不同部位,以580-830 nm段图像更为清晰,530 nm以下图像杂波较多。面部颧、颧下、鼻尖、眉间、额等部位和四指指丘、大小鱼际部位的反光较强,而眼、眉、嘴角和五指末端等则较弱。面部光谱双侧基本对称,而左右手的对称性在不同的个体上不尽相同。在光谱曲线上可以见到某一或者某些波长处光谱出现突变的细节。结论：高光谱成像技术可以清晰地显示人体面和手的图像,显示各部位（器官）的光谱特征;本文得到的面、手高光谱正常数据和特征将为该技术用于疾病诊断和中医辨证提供参考依据。     

In Vitro Identification of Gold Nanorods through Hyperspectral Imaging

Due to their unique plasmonic and optical properties, gold nanorods (GNR) have shown tremendous potential for nano-based applications extending into a variety of fields including bioimaging, sensor development, electronics, and cancer therapy. These distinctive, shape-specific properties are strongly dependent upon the GNR aspect ratio, thus producing the ability to be targeted for an application by fine-tuning their physical parameters. It is owing to their characteristic spectral signature, which is vastly different from that of a cellular setting, that GNRs are emerging as an ideal candidate for nano-based imaging applications. However, one challenge that has emerged in the field of bioimaging is the need to account for the observed plasmon coupling effect that arises from GNR agglomeration in a physiological environment. In this study, GNRs with aspect ratios of 2.5 and 6.0 were actively identified in an in vitro setting through a hyperspectral imaging (HSI) analysis; which successfully recognized and separated the light scattering pattern of these particles from that of the surrounding cells. Through inclusion of agglomerated GNR spectral patterns in the HSI spectral library, this imaging technique was able to overcome the complication of plasmon coupling, though to varying degrees. These results demonstrate the tremendous potential of GNRs coupled with HSI analysis to advance the field of nano-based sensing and imaging mechanisms.     

四棱豆不同生育期叶片中过氧化物酶活力测定

比较了四棱豆不同生育期叶片中过氧化物酶活性,结果表明：该酶活力和比活力在不同生育期存在着明显差异,蛋白质含量变化不大。在成熟期,随着植株节位升高,酶活力及比活力均呈下降趋势,蛋白质含量则呈上升趋势。     

Multicolour Fluorescence Imaging of Sugar Beet Leaves with Different Nitrogen Status by Flash Lamp UV-Excitation

Fluorescence images of leaves of sugar beet plants (Beta vulgaris L. cv. Patricia) grown on an experimental field with different fertilisation doses of nitrogen [0, 3, 6, 9, 12, 15 g(N) m^–2] were taken, applying a new multicolour flash-lamp fluorescence imaging system (FL-FIS). Fluorescence was excited by the UV-range (280–400 nm, _max = 340 nm) of a pulsed Xenon lamp. The images were acquired successively in the four fluorescence bands of leaves near 440, 520, 690, and 740 nm (F₄₄₀, F₅₂₀, F₆₉₀, F₇₄₀) by means of a CCD-camera. Parallel measurements were performed to characterise the physiological state of the leaves (nitrogen content, invert-sugars, chlorophylls and carotenoids as well as chlorophyll fluorescence induction kinetics and beet yield). The fluorescence images indicated a differential local patchiness across the leaf blade for the four fluorescence bands. The blue (F₄₄₀) and green fluorescence (F₅₂₀) were high in the leaf veins, whereas the red (F₆₉₀) and far-red (F₇₄₀) chlorophyll (Chl) fluorescences were more pronounced in the intercostal leaf areas. Sugar beet plants with high N supply could be distinguished from beet plants with low N supply by lower values of F₄₄₀/F₆₉₀ and F₄₄₀/F₇₄₀. Both the blue-green fluorescence and the Chl fluorescence rose at a higher N application. This increase was more pronounced for the Chl fluorescence than for the blue-green one. The results demonstrate that fluorescence ratio imaging of leaves can be applied for a non-destructive monitoring of differences in nitrogen supply. The FL-FIS is a valuable diagnostic tool for screening site-specific differences in N-availability which is required for precision farming.     

Detection and Measurement of Staphylococcus epidermidis Slime Using an ELISA Technique

A polyclonal rabbit anti-serum against the strong slime-producing Staphylococcus epidermidis strain RP62A was absorbed with the slime-negative phase variant of this strain PV1 in order to remove not slime-specific antibodies. Using this antiserum we established an ELISA which enables detection of slime production in S. epidermidis extracts. The ELISA showed high absorbance when extracts from slime-positive strains (confirmed in the tissue culture tube test) were used as antigens. The high absorbance of slime-positive strains was greatly reduced by periodate oxidation of the extracts and was resistant to proteinase digestion suggesting that the detected antigen is composed of polysaccharides. In contrast to other rapid and simple laboratory detection methods for S. epidermidis slime, the slime-specific ELISA gave positive results in the presence of human serum.     

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick’s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.     

Detection of Tephra Layers in Antarctic Sediment Cores with Hyperspectral Imaging

Tephrochronology uses recognizable volcanic ash layers (from airborne pyroclastic deposits, or tephras) in geological strata to set unique time references for paleoenvironmental events across wide geographic areas. This involves the detection of tephra layers which sometimes are not evident to the naked eye, including the so-called cryptotephras. Tests that are expensive, time-consuming, and/or destructive are often required. Destructive testing for tephra layers of cores from difficult regions, such as Antarctica, which are useful sources of other kinds of information beyond tephras, is always undesirable. Here we propose hyperspectral imaging of cores, Self-Organizing Map (SOM) clustering of the preprocessed spectral signatures, and spatial analysis of the classified images as a convenient, fast, non-destructive method for tephra detection. We test the method in five sediment cores from three Antarctic lakes, and show its potential for detection of tephras and cryptotephras.     

不同生长时期多伞阿魏土壤养分及光合特性比较研究

以多伞阿魏(Ferula ferulaeoides)为研究对象,分别对其展叶期、成熟期、果期土壤含水量、pH、有机质、养分和叶片的净光合速率、蒸腾速率、水分利用率等光合指标的日变化进行比较研究。结果表明:土壤含水量、有机质、养分随着多伞阿魏生长呈下降趋势,而pH却有所上升,但变化幅度不大。在不同土壤深度上,土壤含水量、pH随着土层深度的加深逐渐升高,而土壤有机质、养分含量却随土层的加深逐渐减少。多伞阿魏3个时期的净光合速率日变化均为双峰曲线,在14:00出现明显的“光合午休”现象,日平均大小为展叶期>成熟期>果期;蒸腾速率日变化趋势表现为单峰曲线,在中午14:00达到最高值,日平均大小为展叶期>果期>成熟期;水分利用率日变化总体呈上升趋势,在14:00出现最低值,日平均大小为成熟期>果期>展叶期。通过对多伞阿魏净光合速率和蒸腾速率与影响因子的相关分析显示:在整个生长期气孔导度,光合有效辐射与净光合速率呈显著正相关性;光照强度与蒸腾速率有明显正相关性。     

高效液相色谱法测定中国茶中的黄酮醇

本文对五种中国茶中的主要抗氧化物质——黄酮醇的三种单体进行了测定,比较了不同种类茶叶中黄酮醇含量的差异,同时还比较了不同地区绿茶中黄酮醇的含量,并对可能引起这种差异的原因进行了分析。结果表明,茶叶中三种黄酮醇的含量呈现一定规律性,不同种类茶叶中黄酮醇的含量受生产工艺的影响比较大,而不同地区的绿茶中黄酮醇的含量则受日照与培育种类的影响较大。同时,对测定的色谱条件也进行了优化。     

Purification and Properties of Adenosine Nucleosidases from Tea Leaves

Three adenosine nucleosidases (adenosine ribohydrolase, EC 3.2.2.7) with high substrate specificity were isolated from the extracts of tea leaves by a procedure including fractionation with ammonium sulfate, column chromatography on DEAE- and CM-cellulose, and gel filtration on Sephadex G-100. They were designated adenosine nucleosidase I, II and III, respectively, and their properties were characterized.

Among the naturally occurring nucleosides only adenosine and 2′-deoxyadenosine were hydrolyzed by these three enzymes and cleavage rate of the N-glycosidic bond in 2′-deoxyadenosine was three or four times greater than that in adenosine.