The LB Films of Dansyl Chloride Labeled Octadecylamine and Its Fluorescence Lifetime

Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.

The Preliminary Report on Rumen Protozoa Grazing Rate on Bacteria with a Fluorescence-Labeled Technique

Studies on the bacterial predation rate by rumen protozoa were carried out under laboratory conditions using a technique of fluorescence-labeled bacteria （FLB）. Four Xuhuai goats were used in this experiment to obtain rumen protozoa and bacteria. Two groups were designed as follows： One group was the whole bacteria which were labeled using fluorescence through removing free bacteria from rumen fluid （WFLB）; the other group was the bacteria which were labeled using fluorescence without removing free bacteria from rumen fluid （FLB）. The result indicated that the bacterial predation rates of rumen protozoa was 398.4 cells/（cell h） for the group WFLB, 230.4 cells/（cell h） for the group FLB, when the corresponding values expressed as bacteria-N, they were 2.15 pg N/（cell h） for the group WFLB, and 1.24 pg N/（cell h） for the group FLB, respectively. Extrapolating the assimilation quantity of nitrogen by ciliates on bacteria of Xuhuai goat, there were 103.2 mg N/（d capita） for the group WFLB, and 59.5 mg N/（d capita） for the group FLB, respectively. It was estimated that protein losses due to microbial recycling were 0.645 g pro/（d capita） for the group WFLB and 0.372 g pro/（d capita） for the group FLB, respectively. In addition, the fluorescence-labeled technique would be a potential assay for the determination of bacterial predation rate by rumen protozoa.

Preparation, Characterization and Pharmacokinetics of Fluorescence Labeled Propylene Glycol Alginate Sodium Sulfate

A rapid and sensitive fluorescence labeling method was developed and validated for the microanalysis of a sulfated polysaccharide drug,namely propylene glycol alginate sodium sulfate(PSS), in rat plasma. Fluorescein isothiocyanate(FITC) was selected to label PSS, and 1, 6-diaminohexane was used to link PSS and FITC in order to prepare FITC-labeled PSS(F-PSS) through a reductive amination reaction. F-PSS was identified by UV-Vis, FT-IR and 1H-NMR spectrum. The cell stability and cytotoxicity of F-PSS were tested in Madin-Darby canine kidney(MDCK) cells. The results indicated that the labeling efficiency of F-PSS was 0.522% ± 0.0248% and the absolute bioavailability was 8.39%. F-PSS was stable in MDCK cells without obvious cytotoxicity. The method was sensitive and reliable; it showed a good linearity, precision, recovery and stability. The FITC labeling method can be applied to investigating the absorption and metabolism of PSS and other polysaccharides in biological samples.

Application and development of fluorescence probes in MINFLUX nanoscopy(invited paper)

The MINimal emission FLUXes(MINFLUX)technique in optical microscopy,widely recognized as the next innovative fluorescence microscopy method,claims a spatial resolution of 1-3 nm in both dead and living cells.To make use of the full resolution of the MINFLUX microscope,it is important to select appropriate fluorescence probes and labeling strategies,especially in living-cell imaging.This paper mainly focuses on recent applications and developments of fluorescence probes and the relevant labeling strategy for MINFLUX microscopy.Moreover,we discuss the deficiencies that need to be addressed in the future and a plan for the possible progression of MINFLUX to help investigators who have been involved in or are just starting in the field of super-resolution imaging microscopy with theoretical support.

A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity

Objective The assay of DNA mismatch repair （MMR） activity can be used as a biomarker for environmental condition detection and human disease diagnosis. Radioactive 32p-endlabeled DNA containing mismatch is extensively used as the substrate for MMR activity analyses. The aim of the present study is to develop a simple non-radioactive, but equally specific and sensitive method for the MMR activity assay. Methods A fluorescent label was chosen to replace the radioactive isotope label. Sensitive evaluation of the fluorescent label was carried out for the first time, and then the fluorescent label was compared with the isotope label in the MMR activity and DNA binding assays. Result LOD （limit of detection） of the fluorescent label was about 0.1 fmol and the relative signal strength displayed a pretty good linear relationship. Moreover, the fluorescent label method has equivalent sensitivity and performance as compared with the classical radioactive method in experiments. Conclusion In light of the sensitivity, reproducibility, safety, rapidity and long lifespan of the fluorescent label, this improved method can be applied to evaluation of biologic and toxic effects of environmental pollutants on man and other forms of life.

Preparation and Performance of Fluorescein Isothiocyanate⁃Labeled Fluorescent Starch and Polyvinyl Alcohol for Warp Sizing

In order to solve the problems of low accuracy and poor variety adaptability of the current measurement method of the permeability and coating property of sizing paste in textile industry, taking starch and polyvinyl alcohol(PVA) as the representatives of the most commonly used textile sizes, various concentrations of fluorescent molecules fluorescein isothiocyanate(FITC) were used to label starch and PVA to prepare fluorescein(F)-starch and F-PVA fluorescent sizes with different degrees of labeling(DLs) for the first time, respectively. Then the starch and PVA derivatives were employed to size pure cotton warp yarns. After preparing the sections of the sized yarns, the permeability and coating percentage of starch and PVA paste to the yarns were calculated by using a fluorescence microscope and the Photoshop software, respectively. The results demonstrate that F-starch and F-PVA with appropriate DL of 0.791% and 0.161%, respectively, exhibit good fluorescence property and similar sizing performance to the sizing performance of unlabeled starch and PVA. It is considered that fluorescence labeling of sizing agents with fluorescein units can provide an innovative way for the accurate determination of the permeability and coating property of sizing paste to warp yarns.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.

Immunofluorescent Labeling of Human HepG2 Cells with CdTe Quantum Dot Probe Conjugated with Anti-pan CK MAb

A relatively sensitive, specific, and photostable method for the detection of cytokeratin of cancer cells via conjugation with cadmium telluride quantum dots（CdTe QDs） was described. Water soluble CdTe QDs were conjugated to anti-pan-cytokeratin（CK） monoclonal antibody（MAb） through coupling reagent [1-ethyl-3-（3-dimethyla- mino propyl）carbodiimide, EDC] and the conjugates were purified by dialysis. The expression of pan CK protein in HepG2 cells was observed by immunocytochemistry and direct immunofluorescence via QDs-Ab conjugates respectively. Fluorescence intensity and photostability of QDs were compared with those of FITC（fluorescein isothiocyanate）. The results show that the QDs-Ab conjugates recognized specifically pan CK protein in HepG2 cells. Compared with FITC, CdTe QDs had higher brightness and photostability without obvious photobleaching under continuous exciting light illumination for 30 min and after the placement at room temperature for 3 d. The results indicate that conjugates of CdTe quantum dot with anti-pan CK MAb can be used for labeling cancer cells derived from epithelial tissues, which provides the basis for the detection of circulating tumor cells（CTCs）.

Visualization of integrin molecules by fluorescence imaging and techniques

Integrin molecules are transmembraneαβheterodimers involved in cell adhesion,trafficking,and signaling.Upon activation,integrins undergo dynamic conformational changes that regulate their affinity to ligands.The physiological functions and activation mechanisms of integrins have been heavily discussed in previous studies and reviews,but the fluorescence imaging techniques-which are powerful tools for biological studies-have not.Here we review the fluorescence labeling methods,imaging techniques,as well as Förster resonance energy transfer assays used to study integrin expression,localization,activation,and functions.

High Performance Recycling of Polymers by Means of Their Fluorescence Lifetimes

Technical polymers could be identified by means of their remarkably strong auto fluorescence. The time constants of this fluorescence proved to be characteristic for the individual polymers and can be economically determined by integrating procedures. The thus obtained unequivocal identification is presented for their sorting for recycling. Furthermore, polymeric materials were doped with fluorescent dyes allowing a fine-classification of special batches.

荧光RNA及其生物传感技术研究进展

荧光RNA技术是一种新兴的RNA标记技术,可用于活细胞RNA的原位实时标记与成像,对于人们理解RNA的功能和调控机制发挥着至关重要的作用。基于荧光RNA的生物传感技术可用于活细胞内小分子代谢物以及蛋白质等靶标的实时动态检测,为生命科学基础研究以及生物医学传感技术开发提供极具价值的工具。本文对遗传编码的荧光RNA的发展历程、荧光RNA技术在活细胞RNA成像,以及基于荧光RNA的生物传感技术在活细胞代谢物检测等方面的应用进行了介绍和总结,并对该领域的发展现状和未来发展方向展开讨论和展望,以期为该技术的进一步发展和在相关领域的应用提供参考。

Brainbow示踪小鼠心肌细胞增殖中他莫昔芬最适标记剂量的探究

目的:运用Brainbow 2.1小鼠进行心肌细胞谱系追踪,探讨用于心肌细胞增殖标记的最适他莫昔芬剂量。方法:采用4周龄的Brainbowfl/+;Myh6-MerCreMe+小鼠,通过腹腔注射的方式给予3个不同剂量(3、9和27 mg/kg)的他莫昔芬诱导Cre重组酶表达,使心肌细胞标上特定的颜色,4周后取材染色,共聚焦显微镜下定量不同剂量的他莫昔芬标记的心肌细胞数量,以及被标记的细胞中单个细胞、临近的2个细胞簇和临近的3个及以上细胞簇的比例。选择合适剂量(9 mg/kg)的他莫昔芬标记Brainbow小鼠,对比假手术组与心肌梗死(MI)模型组心肌细胞增殖情况。结果:(1)注射9 mg/kg的他莫昔芬后标记的单个细胞比例为99.13%±0.03%,临近的2个细胞簇比例为0.82%±0.09%,3个及以上细胞簇比例为0.05%±0.01%。3 mg/kg组标记总数较少;27 mg/kg组出现明显克隆扩增富集。(2)用9 mg/kg的他莫昔芬标记心肌细胞后,MI小鼠2个细胞簇和3个及以上细胞簇标记比例较假手术组均显著升高(P<0.01),MI后心肌细胞增殖比例增加。结论:9 mg/kg他莫昔芬标记的临近心肌细胞簇的比例更接近生理状态下的心肌细胞增殖比例,因此9 mg/kg为示踪心肌细胞增殖的最适他莫昔芬剂量。

植物乳杆菌WLPL04的荧光标记及其表征

植物乳杆菌WLPL04源自健康母乳,能在肠道定植。对植物乳杆菌WLPL04进行荧光标记,为研究其在体内的定植、移位及分布提供重要实验材料。以pMG36e为骨架,含3种不同启动子(P_(X),P_(23)和P_(ldh))的mCherry红色荧光蛋白基因作为标记基因,构建重组质粒pMG36e-P_(X)-mCherry、pMG36e-P_(23)-mCherry和pMG36e-P_(ldh)-mCherry,转化大肠杆菌,通过PCR、质粒DNA酶切、菌落颜色观察和激光共聚焦显微镜观察进行验证。验证构建成功后进一步将3种重组质粒分别导入植物乳杆菌WLPL04。结果显示导入质粒pMG36e-P_(X)-mCherry、pMG36e-P_(23)-mCherry的植物乳杆菌WLPL04,其菌体在激光共聚焦显微镜下均显示明显红色荧光;而导入pMG36e-P_(ldh)-mCherry的菌体其红色荧光较弱。此外,通过生长曲线和扫描电镜观察分析不同重组质粒对菌株生长及形态的影响,结果表明3种质粒对植物乳杆菌WLPL04的生长及菌体形态没有显著影响。以上结果表明植物乳杆菌WLPL04已被红色荧光蛋白mCherry成功标记,可为后续研究其在体内的定植、移位及分布提供重要实验材料。

基于轻量级卷积神经网络的荧光指示标签用于冷鲜猪肉新鲜度判别

荧光新鲜度标签的颜色指示是实时监测肉制品品质的重要手段。以冷鲜猪肉为研究对象,提出一种由异硫氰酸荧光素(fluorescein isothiocyanate,FITC)、罗丹明B(rhodamine B,RhB)2种荧光素组成的比率型荧光新鲜度指示标签,其中发绿色荧光的FITC为反应信号,发红色荧光的RhB为参考信号。结果表明:当标签与腐败胺反应时,表现出双发射特性,FITC荧光增强,RhB荧光不受干扰,标签呈现红粉色到黄绿色的明显过渡,显著提高了标签的灵敏性和精确性;其次,利用卷积神经网络对荧光新鲜度标签的色泽变化进行智能化判别,以减少人为视觉误差,对比3种轻量级(MobileNetv2、EfficientNetb0、ShuffleNetv2)和2种非轻量级卷积神经网络(ResNet50、VGG16)的判别效果,其中轻量级神经网络EfficientNetb0的效果优于其他4种模型,识别准确率高达95.6%,且参数量和运算量仅为4.01 MB和0.398 GMACs,实现了最佳运算速度和精度的平衡。因此,利用该模型可满足快速、准确、无损判别冷鲜猪肉新鲜度的需求。研究结果可为荧光指示标签应用于冷链物流贮运过程中智能化判别冷鲜猪肉新鲜度提供理论参考。

负载荧光探针的指示标签对三文鱼新鲜度的实时可视化监测

实时可视化监测鱼肉新鲜度,对于保障食品质量和消费者安全至关重要。以二甲氨基肉桂醛和异弗尔酮丙二腈为原料,合成一种具有供体(D)-π-受体(A)结构的荧光探针CDI,用来实时监测三文鱼肉的新鲜度。此探针对13种胺类物质响应明显,且对水产品腐败过程中产生的尸胺的荧光检测限低至9.91μmol/L。采用物理沉降法将探针CDI负载到纤维滤纸上,再加入盐酸使其质子化,得到负载探针CDI+H~+的指示标签。将指示标签置于三文鱼肉上空时,随着三文鱼肉新鲜度的下降,指示标签从白色变为浅黄色直至黄色,荧光逐渐开启,从紫粉色变为橙红色。通过对照总挥发性盐基氮值,验证了负载探针CDI+H^(+)的指示标签可以通过比色荧光双响应信号很好地区分三文鱼肉的3个新鲜度等级。

单病毒示踪技术在动植物细胞中的应用进展

病毒(virus)是一种专营胞内寄生的简单微生物,是造成动植物病害的主要病原体之一。病毒的侵染过程高度动态且复杂,单病毒示踪技术的发展为揭示病毒生命活动的精细过程提供了可能。单病毒示踪是一种基于荧光标记与高时空分辨率成像的新方法,允许对单一或多个病毒的生命活动进行观察。近几年,单病毒示踪技术在病毒入胞、复制、细胞间传染等方面的研究中获得广泛应用,使病毒感染机制的研究取得较大的进展。本文介绍了病毒入侵细胞机制的研究进展,重点总结了荧光标记在单病毒示踪上的应用,特别是对病毒不同结构的标记和成像方法进行了详细论述,以期为优化标记策略、解决对不同种类和结构的病毒实现高效标记等问题提供新思路。

基于量子点标记技术串珠镰孢菌的成像及示踪

本文采用化学腐蚀工艺制备的碳化硅量子点作为新型标记材料,成功实现了其对串珠镰孢菌的标记,并基于该标记技术研究了酚酸类物质对该菌分裂过程的影响,揭示了串珠镰孢菌对拟南芥幼苗的侵染机制。结果表明,腐蚀法制备的量子点直径约为5 nm,近似呈球状,在激发光波长340 nm时光致发光强度峰值达到最大,量子点表面未经修饰既已形成了羟基、羧基及巯基亲有机基团;串珠镰孢菌在苯甲酸的胁迫下呈现出分裂迅速、产孢数量多、孢子侵染毒力强等生长变化态势,且该趋势与苯甲酸含量正相关;长时成像及示踪结果表明,拟南芥根毛区是该菌对其侵染的第一部位,其次为表皮细胞,最后该菌冲破植株最后一道安全屏障细胞壁进入细胞核,完成整个侵染过程。

基于(PEA)_(2)PbBr_(4)单晶的光致发光防伪织物标贴制备与性能

为制备高效轻质的荧光防伪织物标贴,采用反溶剂气相辅助结晶法生长不同尺寸和密度的苯乙胺溴化铅((PEA)_(2)PbBr_(4))单晶,并将其旋涂封装于PVC薄膜内,与织物热轧复合,得到具有光致发光防伪功能的织物标贴;通过XRD、SEM、紫外可见光度计和瞬态荧光光谱仪等测试对单晶的结构、复合膜与织物标贴的发光特性进行分析。结果表明:采用简单温和的工艺即可有效制备晶体并调控其尺寸和密度,制作具有防伪功能的织物标贴。尺寸为5~10μm的二维结构单晶,其厚度约200 nm;通过调节邻二氯苯与N,N-二甲基甲酰胺的溶剂比例能够有效控制(PEA)_(2)PbBr_(4)单晶尺寸。当单晶尺寸约为5μm时,其分布状态、结晶性和发光性能最为优异,其复合膜的吸收峰和发射峰分别位于391 nm和414 nm处。采用超微量的单晶防伪标贴即可在365 nm紫外光的激发下发出强烈均匀的蓝紫色光,并且,用于织物防伪能够达到洗可穿及耐磨标准,实现有效的防伪功能。

SCF泛素连接酶的体外泛素化体系构建与检测

为研究蛋白质的体外泛素化过程,利用大肠杆菌表达系统异源表达和纯化APPBP1/UBA3(E1)、UBC12(E2)、Cullin1-Rbx1(E3)和Nedd8(neural precursor cell expressed developmentally down-regulated protein 8)蛋白,制备FITC-Cysteine绿色荧光素标记的泛素Ub(Ubiquitin),构建了SCF泛素连接酶的体外泛素化体系,同时实现快速检测SCF泛素连接酶中Cullin1蛋白的自泛素化修饰。结果表明,建立的体外SCF E3自泛素化活性反应体系具有较高的可操作性和便利性。