title: Improving fluorescence imaging of biological cells on biomedical polymers.
abstract: Immunofluorescence imaging on polymeric biomaterials is often inhibited by autofluorescence and other optical phenomena. This often limits the analysis that can be performed on cells that are in contact with these materials. This study outlines a method that will quench these inhibitive optical phenomena on a variety of polymeric materials, including poly(glycerol sebacate), poly(urethane), poly(L-lactide-co-ε-caprolactone), and poly(lactic acid-co-glycolic acid). The method uses a simple material treatment method utilizing Sudan Black B (SB), which is commonly used as an autofluorescence quenching molecule in tissue histology, but has not yet been used in biomaterials analysis. The quenching mechanism in the selected polymers is investigated using attenuated total reflectance Fourier transform infrared spectroscoy, ultraviolet-visible light absorbance and fluorescence analysis, and scanning electron microscopyobservation of the material morphology prior to and after SB treatment. The results point to SB eliminating the inhibitive light phenomena of these materials by two methods: (i) chemical interaction between SB and the polymer molecules and (ii) physical interaction whereby SB forms a physical barrier that can absorb scattered light and quench autofluorescence interference during fluorescence microscopy. The studies show that the treatment of polymers with SB is robust across the polymers tested, in both porous and non-porous formats. The method does not interfere with immunofluorescent imaging of fluorescently labeled biological cells cultured on these polymers. This quick, simple, and affordable method enables a variety of analyses to be conducted that may otherwise have been impractical or impossible.
authors.affiliation: Department of Mechanical Engineering and Mechanics, Lehigh University, Bethlehem, PA 18015, USA.
authors.forename: Israd H
authors.initials: IH
authors.lastname: Jaafar
authors.forename: Courtney E
authors.initials: CE
authors.lastname: LeBlon
authors.forename: Ming-Tzo
authors.initials: MT
authors.lastname: Wei
authors.forename: Daniel
authors.initials: D
authors.lastname: Ou-Yang
authors.forename: John P
authors.initials: JP
authors.lastname: Coulter
authors.forename: Sabrina S
authors.initials: SS
authors.lastname: Jedlicka
mesh_terms: D001391:Azo Compounds / Q000737:chemistry; D001672:Biocompatible Materials / Q000737:chemistry / Q000494:pharmacology*; D002478:Cells, Cultured; D003651:Decanoates / Q000737:chemistry / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005990:Glycerol / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D019344:Lactic Acid / Q000737:chemistry / Q000494:pharmacology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence / Q000379:methods*; D009281:Naphthalenes; D011100:Polyglycolic Acid / Q000737:chemistry / Q000494:pharmacology; D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011108:Polymers / Q000737:chemistry / Q000494:pharmacology*; D011140:Polyurethanes / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D001391:Azo Compounds; D001672:Biocompatible Materials; D003651:Decanoates; D009281:Naphthalenes; D011108:Polymers; D011140:Polyurethanes; C469892:poly(glycerol-sebacate); D000077182:Polylactic Acid-Polyglycolic Acid Copolymer; D011100:Polyglycolic Acid; D019344:Lactic Acid; C016118:Sudan Black B; D005990:Glycerol