In vivo quantification of fluorescent molecular markers in real-time: A review to evaluate the performance of five existing methods

With the advent of molecular-targeted fluorescent markers, there is a renewed interest in fluorescence quantification methods that are based on continuous wave excitation and multi-spectral image acquisition. However, little is known about their in vivo quantification performance. We reviewed the performance of five selected methods by analytically describing these and varying input parameters of irradiance, excitation geometry, collection efficiency, autofluorescence, melanin content, blood volume, blood oxygenation and tissue scattering using optical properties representing those for human skin. We identified one method that corrects for variations in all parameters. This requires image acquisition before and after marker administration, under identical geometry. Hence, it is suited for applications where the site of interest can be relocated (e.g. anaesthetized animals and dermatology). For applications where relocation is not possible, we identified a second method where the uncertainty in the fluorescence signal was ±20%. Hence, use of these methods can substantially aid in vivo fluorescence quantification compared to use of the raw fluorescence signal, as this changed by more than 3 orders of magnitude. Since these methods can be computed in real-time, they are of particular interest for applications where direct feedback is critical, as diagnostic screening or image-guided surgery.     

银染端粒重复序列扩增法定量检测端粒酶活性的研究

目的：探讨运用简便、快速及无放射性污染的银染粒重复序列扩增法（silver staining telomeric repeat amplification protocol,SS-TRAP）定量检测端粒酶活性。方法：运用SS－TRAP法检测了293细胞、经加热处理的293细胞、空白对照及典型乳腺癌组织、良性乳腺病变标本中端粒酶活性，其结果予以定量。结果：该法有检测出10个以上293细胞中端粒酶活性，热处理及空白对照均为阴性；乳癌组织标本中银染条带清晰可见，而良性病变中未见端粒酶阳性条带。结论：非核素银染TRAP法可用于端粒酶活性定量检测，与TRAP法相比，同样具有较高敏感性和特异性，但更快速、简便。     

Scatter correction in scintigraphy: the state of the art

In scintigraphy, the detection of scattered photons degrades both visual image analysis and quantitative accuracy. Many methods have been proposed and are still under investigation to cope with scattered photons. The main features of the problem of scattering in radionuclide imaging are presented first, to provide a sound foundation for a critical review of the existing scatter correction techniques. These are described using a classification relating to their aims and principles. Their theoretical potentials are analysed, as well as the difficulties of their practical implementation. Finally, the problems of their evaluation and comparison are discussed. Correspondence to: I. Buvat     

中药药性量化方法对补虚药功效归类预测的研究

目的对中药药性依其重要性进行更深层次的多值量化分析,将比定性的文字表述和简单的二值量化表示更有意义.方法以补虚药为研究对象,药性为基本特征,在对其进行二值量化和多值量化的基础上,采用应用人工神经网络和决策树等数据库知识发现技术,进行对补虚药功效归类判别结果的影响研究.结论结果表明药性的不同量化方法对补虚药的功效归类预测有一定影响,多值量化比二值量化具有更为理想的判别结果.     

用显微分光光度计对几种不同类型肺癌癌细胞DNA含量的初步观测

本文用显微分光光度计对39例手术切除的不同组织学类型的人体肺癌标本癌细胞核的DNA含量进行了测量。其中高、中、低分化的鳞癌分别为3、6和5例;高、中、低分化的腺癌分别为3、5和5例;未分化癌大细胞型和小细胞型各为3例和6例。结果发现各类型肺癌的DNA含量均有统计学上的差异(P<0.01)。各DNA含量直方图的峰值部位及分布范围亦不同。从而提示了根据DNA含量的不同,可能有助于肺癌组织学类型及其分化程度的判别:而DNA含量直方图对肺癌类型的判别亦可能是有益的。     

实时定量RT-PCR的原理及方法

实时定量RT-PCR广泛应用于定量检测mRAN表达水平,为基础研究、分子药物学和生物技术研究提供了一种有力的方法。该法具有易操作、高通量、敏感性高和特异性强的特点,随着新酶、新探针和新仪器的发展而得到快速的发展。本文将对实时定量RT-PCR的定量原理、仪器应用、探针种类的研究进展以及细胞因子mRNA表达水平检测上的应用作一综述。     

5′-Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma

 DNA methylation plays an important part in the regulation of gene expression. Alterations in DNA methylation in tumours have been reported and have been used to generate hypotheses about mutagenesis and silencing of tumour suppressor genes. However, the underlying mechanism is still poorly understood, and conflicting data on the levels of overexpression of 5′-cytosine DNA methyltransferase in sporadic colon carcinoma have been published. We used a competitive RT-PCR assay for quantification of mRNA of 5′-cytosine DNA methyltransferase in colon biopsies obtained from patients with hereditary colon carcinoma syndromes and compared the results with those obtained in a control group. No significant difference was found between the flat mucosa of FAP patients and the mucosa of the control group. In FAP and HNPCC patients, the 5′-cytosine DNA methyltransferase mRNA levels of adenomas were significantly higher (P<0.05) than of flat mucosa in the same group, but both showed great variability from patient to patient. Our findings suggest that the mRNA levels of methyltransferase cannot be used as predictive marker for screening in families affected by hereditary colon carcinoma. Received: 20 July 1998 / Accepted: 21 September 1998     

Quantification of SARS-CoV-2 spike and nucleocapsid proteins using isotope dilution tandem mass spectrometry

The emergence and subsequent global outbreak of the novel coronavirus SARS-CoV-2 prompted our laboratory to launch efforts to develop methods for SARS-CoV-2 antigen detection and quantification. We present an isotope dilution mass spectrometry method (IDMS) for rapid and accurate quantification of the primary antigens, spike and nucleocapsid proteins. This IDMS method utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze sample tryptic digests for detection and quantification of selected conserved peptides of SARS-CoV-2 spike and nucleocapsid proteins. The IDMS method has the necessary attributes to be successfully utilized for accurate quantification in SARS-CoV-2 protein-based vaccines and as targets of rapid diagnostic tests. Absolute quantification was achieved by quantifying and averaging 5 peptides for spike protein (3 peptides in the S1 subunit and 2 peptides in the S2 subunit) and 4 peptides for nucleocapsid protein. The overall relative standard deviation of the method was 3.67% for spike protein and 5.11% for nucleocapsid protein. IDMS offers speed (5 h total analysis time), sensitivity (LOQ; 10 fmol/µL) and precision for quantification of SARS-CoV-2 spike and nucleocapsid proteins.     

Quantification and preservation of lectin binding by isolated cardiomyocytes

During preparation of cells for experimentation a considerable amount of bound substance is lost. Our aim was to develop a protocol which retained lectin binding to an extent similar to living cells. This procedure would use fixation procedures suited for fluorescent lectin conjugates and gold-conjugates to be visualized by light- and electron microscopy, respectively. We tested glutaraldehyde and paraformaldehyde in different concentrations before and after lectin binding, different buffers and divalent cations, as additives, to determine the effects on preservation of lectin binding. Lectin binding was visualized and semiquantitatively evaluated by image analysis in the light microscope after silver enhancement of lectin-gold conjugates and by using tetramethyl rhodaminyl isothiocyanate (TRITC)-conjugated lectins. Preservation of lectin binding was best visualized with fluorescent lectin conjugates, whereas during silver enhancement procedures of gold-conjugated lectins, a considerable amount of bound lectins was lost. In general, lectin binding to living cells followed by fixation is superior to fixation before lectin binding. Unfavourable combinations of fixatives and buffers can cause a loss of more than 90% bound lectin. In our experiments with freshly isolated guinea pig cardiomyocytes, lectin binding was best when we used Na-cacodylate buffer with glutaraldehyde fixation (0.1%) after binding of lectins to the living cells.     

Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique

This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed, and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of ‘edges’ present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.