J C Stockert, E N Durantini, E J Gonzalez Lopez, J E Durantini, A Villanueva, R W Horobin
{"title":"阳离子光敏剂ZnTM2、3PyPz对活细胞线粒体的荧光标记,以及氧化还原过程和假碱形成在促进染料摄取中的可能作用。","authors":"J C Stockert, E N Durantini, E J Gonzalez Lopez, J E Durantini, A Villanueva, R W Horobin","doi":"10.1080/10520295.2022.2090603","DOIUrl":null,"url":null,"abstract":"<p><p>The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.</p>","PeriodicalId":8970,"journal":{"name":"Biotechnic & Histochemistry","volume":null,"pages":null},"PeriodicalIF":1.6000,"publicationDate":"2022-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"2","resultStr":"{\"title\":\"Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake.\",\"authors\":\"J C Stockert, E N Durantini, E J Gonzalez Lopez, J E Durantini, A Villanueva, R W Horobin\",\"doi\":\"10.1080/10520295.2022.2090603\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.</p>\",\"PeriodicalId\":8970,\"journal\":{\"name\":\"Biotechnic & Histochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.6000,\"publicationDate\":\"2022-10-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biotechnic & Histochemistry\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1080/10520295.2022.2090603\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2022/7/19 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnic & Histochemistry","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1080/10520295.2022.2090603","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2022/7/19 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake.
The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.
期刊介绍:
Biotechnic & Histochemistry (formerly Stain technology) is the
official publication of the Biological Stain Commission. The journal has been in continuous publication since 1926.
Biotechnic & Histochemistry is an interdisciplinary journal that embraces all aspects of techniques for visualizing biological processes and entities in cells, tissues and organisms; papers that describe experimental work that employs such investigative methods are appropriate for publication as well.
Papers concerning topics as diverse as applications of histochemistry, immunohistochemistry, in situ hybridization, cytochemical probes, autoradiography, light and electron microscopy, tissue culture, in vivo and in vitro studies, image analysis, cytogenetics, automation or computerization of investigative procedures and other investigative approaches are appropriate for publication regardless of their length. Letters to the Editor and review articles concerning topics of special and current interest also are welcome.