title: Synthesis and Characterization of 8-O-Carboxymethylpyranine (CM-Pyranine) as a Bright, Violet-Emitting, Fluid-Phase Fluorescent Marker in Cell Biology.
abstract: To avoid spectral interference with common fluorophores in multicolor fluorescence microscopy, a fluid-phase tracer with excitation and emission in the violet end of the visible spectrum is desirable. CM-pyranine is easily synthesized and purified. Its excitation and emission maxima at 401.5 nm and 428.5 nm, respectively, are well suited for excitation by 405-nm diode lasers now commonly available on laser-scanning microscopes. High fluorescence quantum efficiency (Q = 0.96) and strong light absorption (ε405 > 25,000 M-1cm-1) together make CM-pyranine the brightest violet aqueous tracer. The fluorescence spectrum of CM-pyranine is invariant above pH 4, which makes it a good fluid-phase marker in all cellular compartments. CM-pyranine is very photostable, is retained for long periods by cells, does not self-quench, and has negligible excimer emission. The sum of its properties make CM-pyranine an ideal fluorescent tracer. The use of CM-pyranine as a fluid-phase marker is demonstrated by multicolor confocal microscopy of cells that are also labeled with lipid and nuclear markers that have green and red fluorescence emission, respectively.
authors.affiliation: Center for Biomedical Engineering and Technology, and Department of Physiology, University of Maryland School of Medicine, 111 South Penn Street, Baltimore, Maryland 21201, United States of America.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Legenzov
authors.affiliation: Center for Biomedical Engineering and Technology, and Department of Physiology, University of Maryland School of Medicine, 111 South Penn Street, Baltimore, Maryland 21201, United States of America.
authors.forename: Nathaniel D A
authors.initials: ND
authors.lastname: Dirda
authors.affiliation: Center for Biomedical Engineering and Technology, and Department of Physiology, University of Maryland School of Medicine, 111 South Penn Street, Baltimore, Maryland 21201, United States of America.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hagen
authors.affiliation: Center for Biomedical Engineering and Technology, and Department of Physiology, University of Maryland School of Medicine, 111 South Penn Street, Baltimore, Maryland 21201, United States of America.
authors.forename: Joseph P Y
authors.initials: JP
authors.lastname: Kao
mesh_terms: D065638:Absorption, Radiation; D000818:Animals; D001190:Arylsulfonates / Q000138:chemical synthesis* / Q000494:pharmacology; D002460:Cell Line; D002522:Chlorocebus aethiops; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000494:pharmacology / Q000528:radiation effects; D011721:Pyrenes / Q000138:chemical synthesis* / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: C000607380:8-O-carboxymethylpyranine; D001190:Arylsulfonates; D005456:Fluorescent Dyes; D011721:Pyrenes