Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response

Electrospray ionization-mass spectrometry (ESI-MS) is a very promising tool for the analysis of phospholipid compositions, but is hampered by the fact that not all molecular species are detected with equal efficiency. We studied this and other issues that need to be taken into account to obtain truly quantitative compositional data. The key findings were as follows: First, the instrument response for both saturated and unsaturated phospholipid species decreased with increasing acyl chain length. This effect became increasingly prominent with increasing overall lipid concentration. Second, the degree of acyl chain unsaturation also had a significant effect on instrument response. At the highest concentration studied (10 pmol/microl), polyunsaturated species gave 40% higher intensity than the fully saturated ones. The effect of unsaturation diminished and nearly disappeared with progressive dilution. Third, the instrument response for the different head group classes varied markedly depending on the infusion solvent used. Notably, inclusion of ammonia in the infusion solvent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the spectra. The fact that instrument response is dependent on many structural features, overall lipid concentration, solvent composition, and instrument settings makes it necessary to include several internal standards for each phospholipid class to obtain accurate data. Preferably, both unsaturated and saturated standards should be used. Finally, we quantified the major phospholipid classes of BHK cells using ESI-MS. The data agreed closely with those obtained with thin-layer chromatography and phosphorus analysis. This study indicates that quantitative compositional data can be obtained with ESI-MS, provided that proper attention is paid to experimental details, particularly the choice of internal standards.     

The effects of pulsed, high frequency radio waves on rat liver (ultrastructural and biomedical observations)

The effects of a high frequency electro-magnetic field, generated by a Diapulse instrument (Diapulse Corporation of America) on rat liver has been investigated. Ultrastructural aspects are described and quantitative determinations of mitochondrial enzymes MAO, CyT-Ox, MDH, SDH and ATP-ase recorded. The standard therapeutic parameters generally used with the Diapulse instrument in medicine were found to induce a stimulation effect at the investigated level, without apparent degenerative modifications. A concordance between the qualitative ultrastructural data and quantitative subcellular enzymic determinations has been observed.     

Qualitative und quantitative Fluoreszenzmikrospektrographie mit dem Leitz-Mikrospektrographen

Zusammenfassung Ausgehend vom Leitz-Mikrospektrographen wird gezeigt, wie dieses Gerät für mikrospektrofluorimetrische Untersuchungen und für die quantitative Fluoreszenzcytophotometrie ausgerüstet werden kann. Die im Zusammenhang mit der Anwendung des Gerätes auftretenden Fragen der Eichung und Korrektur der Spektralkurven werden erörtert.

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Qualitative and quantitative fluorescence cytophotometry at the Leitz-Microspectrograph

Summary The use and additional equipment of the Leitz-Mikrospectrograph for microspectrofluorometry and fluorescence cytophotometry are described in detail.Fluorescence excitation with incident light together with the use of dichromatic beam splitters for optimum separation of the excitation beam from emitted radiation are the characteristic features of this instrument.The fluorescence light is spectrally dispersed by a glass-prism (Special glass for visible light, quarz for UV).A motor driven oscillating mirror moves along the spectrum and reflects the light of continuously changing wavelength on to the photo-cathode of the multiplier, that transfers the optical impulse into an electrical signal. The wavelength dependend change of spectral intensity is registered by a light beam oscillograph fitted with four fixed potentiometers indicating the 400, 500, 600 and 700 nm wavelength position by crosspoints with an oblique line drawn by the wavelength potentiometer, while the sixth potentiometer records the spectral curve.Problems of spectral calibration and correction are discussed.When the prism and the oscillating mirror are removed the instrument may be used as fluorescence cytophotometer. For this purpose of quantitative fluorescence cytophotometry a digital measuring unit with a print-out instrument is presented.

     

嵌入式AFP测试仪的设计

介绍了一个嵌入式AFP测试仪的设计。测试仪以嵌入式微处理器LPC2210为核心,主要包括数据采集、步进电机的驱动及人机交互界面等几部分。测试仪可通过RS232与PC机通讯,进行数据处理及报告分析,具有一定的临床辅助诊断价值。     

A high throughput, interactive imaging, bright-field wound healing assay

The wound healing assay is a commonly used technique to measure cell motility and migration. Traditional methods of performing the wound healing assay suffer from low throughput and a lack of quantitative data analysis. We have developed a new method to perform a high-throughput wound healing assay that produces quantitative data using the LEAP? instrument. The LEAP? instrument is used to create reproducible wounds in each well of a 96-well plate by laser ablation. The LEAP? then records bright field images of each well at several time points. A custom texture segmentation algorithm is used to determine the wound area of each well at each time point. This texture segmentation analysis can provide faster and more accurate image analysis than traditional methods. Experimental results show that reproducible wounds are created by laser ablation with a wound area that varies by less than 10%. This method was tested by confirming that neuregulin-2β increases the rate of wound healing by MCF7 cells in a dose dependent manner. This automated wound healing assay has greatly improved the speed and accuracy, making it a suitable high-throughput method for drug screening.     

Quantitative gas chromatographic determination of 3-methylhistidine in biological fluids

The gas chromatographic procedure is suggested to determine 3-methylhistidine in biological fluids. The amino acid fraction containing 3-methylhistidine is separated by ion-exchange chromatography. Amino acids are transformed into N-trifluoroacetyl-O-isobutyl esters which are analyzed by the gas chromatography instrument with micropacked columns and ionization-resonance detector. The limit of the quantitative determination of 3-methylhistidine is 50 ng per a probe.     

High‐performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis: Observations and implications

We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods on both instruments, namely label‐free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3‐based reporter ion isolation Synchronous Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label‐free approach offers a more linear response with a wider dynamic range than MS/MS‐based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We observed, however, that the choice of quantitative approach had little impact on the ability to statistically evaluate the E. coli heat shock response. We conclude that in the experimental conditions tested, MS/MS‐based reporter ion quantitation provides reliable biological insight despite the issue of compressed dynamic range, an observation that significantly impacts the choice of instrument.     

阿达玛变换显微图象法测定乳腺癌DNA含量

以吖啶橙为细胞DNA荧光探针,用阿达玛(Hadamard)变换显微图象分析仪和显微荧光光度计分别测定了4例乳腺肿瘤的细胞DNA含量(倍性),并对分析结果进行了比较,二者的分析结果均与病理学诊断结论相吻合.阿达玛变换显微图象分析仪作为一种新的细胞定量分析仪器,其分析结果的准确度可与显微荧光光度计相媲美,而且阿达玛变换显微图象分析仪还具有其独特的优点,如信噪比高、具有同时分析多个细胞和同步扣除背景信号的能力等.     

Single-stranded conformational polymorphism analysis using automated capillary array electrophoresis apparatuses

We describe a new environment of a single-stranded conformational polymorphism (SSCP) analysis using automated capillary array sequencers (e.g., ABI Prism 3100 and 3700). In this environment, electrophoretic conditions, settings for instrument management, and software for data analysis are adjusted for SSCP analysis. Highly reproducible results are obtained with this new system, and fragments with mutations and/or polymorphisms in different capillaries or different runs can be reliably detected. The relative peak heights between alleles are quantitative and reproducible between runs, and so allele frequencies of single nucleotide polymorphisms can be accurately estimated by a pooled DNA strategy. The method allows unattended, low-cost, and quantitative SSCP analysis using instruments that are widely accessible.     

Evaluating the palatability of fermented foods

ABSTRACT

The present review deal with the novel studies which present possibility of generating a novel sensory evaluation instrument for describing comprehensive food palatability. These studies suggest the palatability can be dissected into its componential subdomains and the subdomains in turn reconstitute comprehensive palatability with evaluation of the quantitative contribution of each subdomain. The studies suggest the novel instrument is useful for comprehensive evaluation of palatability of multifarious fermented foods.     

Continuous measurement of glucose utilization in heart myoblasts

Understanding quantitative aspects of cell energy metabolism and how it is influenced by environment is central to biology, medicine, and biotechnology. Most methods used for measuring metabolic fluxes associated with energy metabolism require considerable personnel effort or high maintenance instrumentation. The microphysiometer is a commercially available instrument that measures acid extrusion rates, which are commonly used for drug screening. With the addition of oxygen sensors, the instrument can also be used to measure cell oxygen consumption rates and thereby calculate glycolytic fluxes. In the work described here, oxygen consumption and acid extrusion rates were used to measure glucose utilization by the H9c2 rat heart myoblast cell line and these results are compared with fluxes measured with a radiometric assay. Both assays were used to investigate changes in H9c2 energy metabolism due to cell stimulation with carbachol and insulin. The results demonstrate the utility of the microphysiometer method for measuring both transient and sustained changes in partitioning of glucose utilization between glycolysis and oxidation in live cells.     

An automated, handheld biosensor for aflatoxin

A new immunoaffinity fluorometric biosensor has been developed for detecting and quantifying aflatoxins, a family of potent fungi-produced carcinogens that are commonly found in a variety of agriculture products. They have also been cited as a biological agent under weapons development. The handheld, self-contained biosensor is fully automatic, highly sensitive, quick, quantitative, and requires no special storage. Approximately 100 measurements can be made before refurbishment is required, and concentrations from 0.1 parts per billion (ppb) to 50 ppb can be determined in <2 min with a 1 ml sample volume. The device operates on the principles of immunoaffinity for specificity and fluorescence for a quantitative assay. The analytic procedure is flexible so that other chemical and biological analytes could be detected with minor modifications to the current device. Advances in electro-optical components, electronics, and miniaturized fluidics were combined to produce this reliable, small, and versatile instrument.     

Changes in erythrocyte velocity in microvessels measured with a microprismatic grating

Using an instrument designed by the Ernst Leitz Wetzlar Company (FRG) red blood cell velocity was evaluated in model and intravital experiments. The instrument provides for an accurate quantitative determination of blood microflow velocity, enables the measurements within a wide range of velocities and diameters of microvessels. It is more unsophisticated as regards the design and operation than other instruments intended for similar purposes.     

An optical platform for cell tracking in adult zebrafish

Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research.     

新定量PCR数据处理方法的理论探讨

日新月异的生命科学技术的发展及临床医学科学研究的需求,一般的PCR技术已远远不能满足工作的需要。PE公司在进行了大量的PCR动力学研究的基础上,发现了利用荧光标记探针在PCR循环过程中积累的荧光强度达到仪器捡出阈值时,系统的初始模板数量与循环次数之间有线性关系,据此建立了目前的PE 7700 、PE 5700仪器的定量PCR技术,开创了PCR技术的新局面。但是由于这一技术的误差较大,尚不能满足生命科学及临床医学科学研究的需求,因此需要继续研究新的定量PCR技术。PCR动力学数学模型是根据PCR 技术的原理提出的,能够准确描述PCR反应产物分子数量积累规律的动力学方程,给出了PCR产物数量或者荧光强度与初始模板数量及其他反应条件间的函数关系。利用这一关系,根据PCR反应已积累的产物数量,可以实现准确的定量PCR分析,得到初始模板数量达到定量PCR的目的。使用动力学数学模型做定量PCR分析,其结果的误差仅与使用的荧光强度数值的精确度相关。使用精确到6位数的荧光强度数据,模板数自100~1 000 000区间定量结果的准确性可达99%以上。本文根据模拟实验数据进行了初步的定量PCR分析,结果提示,目前的定量PCR仪器使用PCR动力学模型理论处理分析数据,定量分析的结果会比目前的CT值方法在准确性方面提高几十倍以上,可以满足各方面研究工作误差水平的需要。 Abstract:Today standard PCR can't satisfy the need of biotechnique development and clinical research any more.After numerous dynamic research,PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700.But the error of this technique is too great to satisfy the need of biotechnique development and clinical research.A better quantitative PCR technique is needed.The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system.This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions,and can reflect the accumulating rule of PCR product molecule accurately.Accurate quantitative PCR analysis can be made use this function relation.Accumulated PCR product quantity can be obtained from initial template number.Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used.For an example,when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1 000 000,the quantitative result accuracy will be more than 99%.The difference of result error is distinct using same condition,same instrument but different analysis method.Moreover,if the PCR quantitative analysis system is used to process data,it will get result 80 times of accuracy than using CT method.     

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。     

A semiautomatic instrument for the radioautographic coating technique.

By means of a mechanical coating instrument a fast, simple method to coat specimens with liquid nuclear track emulsion has been devised for quantitative light and electron microscopic radioautography. In both cases, the section is mounted on a glass slide. After the vertically held slide has been immersed in the melted emulsion, the instrument withdraws it at a slow, constant speed. As a result, the specimen is coated with a thin, uniform emulsion layer composed of homogeneously distributed silver bromide crystals. The thickness of the emulsion coat may be standardized by selection of an optimal combination of emulsion dilution, temperature and withdrawal speed.     

Research Progress of Raman Spectroscopy in Drug Analysis

Raman spectroscopy is a spectroscopic analysis technique that enables rapid qualitative and quantitative detection based on inelastic collision and Raman scattering intensity. This review detailed the generation principle, instrument composition, influencing factors, and common classifications of Raman spectrum. Furthermore, it summarized and forecast the research progress of Raman spectroscopy in the field of drug analysis simultaneously over the past decade, including the identification of active pharmaceutical ingredients (APIs), qualitative and quantitative studies of pharmaceutical preparations, detection of illicit drugs, the identification of Chinese herbal medicines, and the combination with other technologies. The development of Raman spectroscopy in other fields is additionally summarized.