title: Widefield imaging of upconverting nanoparticles on epifluorescence microscopes adapted for laser illumination with top-hat profile.
abstract: We describe a modification of epifluorescence microscopes that allows quantitative widefield imaging of samples labeled by upconverting nanoparticles (UCNP). A top-hat illumination profile on the sample was achieved with a 980-nm laser diode by using tandem microlens arrays, a moving diffuser and a telescope, which adjusts the top-hat area to the field of view. Illumination homogeneity is a critical factor for imaging of UCNP since the intensity of their luminescence typically scales with the second power of the excitation intensity. Our illuminator is combined with the epifluorescence attachment of the microscope, allowing easy switching between observation of UCNP and traditional fluorescent dyes. Illumination profile homogeneity of about 98% was measured for objectives with magnification from 4× to 100×, and the top-hat profile was also obtained with phase contrast objectives. We demonstrate capability of the illuminator by evaluating in vitro uptake of UCNP encapsulated in oleyl-hyaluronan micelles into breast cancer cells. Micelles bearing the targeting peptide were about an order of magnitude more efficient than nontargeted micelles.
authors.affiliation: Charles University in Prague, Department of Medical Biophysics and Informatics, Third Faculty of Medicine, Ruska 87, 100 00 Prague 10, Czech RepublicbContipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Mrazek
authors.affiliation: Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Pospisilova
authors.affiliation: Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Vit
authors.initials: V
authors.lastname: Svozil
authors.affiliation: Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Cadek
authors.affiliation: Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Kristina
authors.initials: K
authors.lastname: Nesporova
authors.affiliation: Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Romana
authors.initials: R
authors.lastname: Sulakova
authors.affiliation: Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Brandejsova
authors.affiliation: Charles University in Prague, Department of Medical Biophysics and Informatics, Third Faculty of Medicine, Ruska 87, 100 00 Prague 10, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Vranova
authors.affiliation: Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Velebny
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D006801:Humans; D007834:Lasers; D008029:Lighting; D049449:Luminescence; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D053758:Nanoparticles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D005456:Fluorescent Dyes