title: Evaluation and optimization of multiple fluorophore analysis of a Pseudomonas aeruginosa biofilm.
abstract: Conventional laser scanning microscopy for multiple fluorescent stains can be a useful tool if the problems of autofluorescence and cross-talk are eliminated. The technique of spectral imaging was employed to unmix five different fluorophores - ranging in emission from 435 to 665 nm - applied to a Pseudomonas aeruginosa biofilm with overlapping spectra and which was not possible using traditional channel mode operation. Using lambda scanning and linear unmixing, the five fluorophores could be distinguished with regions of differentiation apparent.
authors.affiliation: University of Vermont, School of Engineering, Burlington, Vermont 05405-0156, USA.
authors.forename: Fiona J
authors.initials: FJ
authors.lastname: Baird
authors.forename: Marilyn P
authors.initials: MP
authors.lastname: Wadsworth
authors.forename: Jane E
authors.initials: JE
authors.lastname: Hill
mesh_terms: D001557:Benzenesulfonates / Q000737:chemistry; D018441:Biofilms; D002138:Calibration; D003208:Concanavalin A / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000166:cytology / Q000502:physiology*; D012235:Rhodamines / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D001557:Benzenesulfonates; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D009942:Organometallic Compounds; D012235:Rhodamines; C103389:SYTO 9; C515601:Sypro Ruby; D003208:Concanavalin A; C005358:tetramethylrhodamine; C007061:C.I. Fluorescent Brightening Agent 28; D016650:Fluorescein-5-isothiocyanate