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.

Charles University Prague Department of Medical Biophysics and Informatics 3rd Faculty of Medicine Ruska 87 100 00 Prague 10 Czech Republic

Charles University Prague Department of Medical Biophysics and Informatics 3rd Faculty of Medicine Ruska 87 100 00 Prague 10 Czech RepublicbContipro Biotech Dolni Dobrouc 401 Dolni Dobrouc 561 02 Czech Republic

Contipro Biotech Dolni Dobrouc 401 Dolni Dobrouc 561 02 Czech Republic

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