Research Papers: Imaging

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

[+] Author Affiliations
Jiri Mrazek

Charles University in Prague, Department of Medical Biophysics and Informatics, Third Faculty of Medicine, Ruska 87, 100 00 Prague 10, Czech Republic

Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic

Martina Pospisilova, Vit Svozil, Ondrej Cadek, Kristina Nesporova, Romana Sulakova, Martina Brandejsova, Vladimir Velebny

Contipro Biotech, Dolni Dobrouc 401, Dolni Dobrouc 561 02, Czech Republic

Jana Vranova

Charles University in Prague, Department of Medical Biophysics and Informatics, Third Faculty of Medicine, Ruska 87, 100 00 Prague 10, Czech Republic

J. Biomed. Opt. 21(5), 056007 (May 27, 2016). doi:10.1117/1.JBO.21.5.056007
History: Received January 31, 2016; Accepted May 2, 2016
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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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© 2016 Society of Photo-Optical Instrumentation Engineers

Citation

Jiri Mrazek ; Martina Pospisilova ; Vit Svozil ; Ondrej Cadek ; Kristina Nesporova, et al.
"Widefield imaging of upconverting nanoparticles on epifluorescence microscopes adapted for laser illumination with top-hat profile", J. Biomed. Opt. 21(5), 056007 (May 27, 2016). ; http://dx.doi.org/10.1117/1.JBO.21.5.056007


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