Widefield imaging of upconverting nanoparticles on epifluorescence microscopes adapted for laser illumination with top-hat profile

Jiri Mrazek; Martina Pospisilova; Vit Svozil; Ondrej Cadek; Kristina Nesporova; Romana Sulakova; Martina Brandejsova; Jana Vranova; Vladimir Velebny

doi:10.1117/1.JBO.21.5.056007

27 May 2016 Widefield imaging of upconverting nanoparticles on epifluorescence microscopes adapted for laser illumination with top-hat profile

Jiri Mrazek, Martina Pospisilova, Vit Svozil, Ondrej Cadek, Kristina Nesporova, Romana Sulakova, Martina Brandejsova, Jana Vranova, Vladimir Velebny

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Journal of Biomedical Optics, Vol. 21, Issue 5, 056007 (May 2016). https://doi.org/10.1117/1.JBO.21.5.056007

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.

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Jiri Mrazek, Martina Pospisilova, Vit Svozil, Ondrej Cadek, Kristina Nesporova, Romana Sulakova, Martina Brandejsova, Jana Vranova, and Vladimir Velebny "Widefield imaging of upconverting nanoparticles on epifluorescence microscopes adapted for laser illumination with top-hat profile," Journal of Biomedical Optics 21(5), 056007 (27 May 2016). https://doi.org/10.1117/1.JBO.21.5.056007

Published: 27 May 2016

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