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.

Lengthening the Lifetime of Common Emissive Probes to Microseconds by a Jigsaw‐Like Construction of NIR‐Responsive Nanohybrids

“…In addition, luminescent probes usually exhibit low photostability as well as fast photobleaching, and absorb in the ultraviolet‐visible spectral region, thus limiting imaging depth. [ 1–5 ] Multiphoton excitation enables near infrared (NIR)‐activation of many well‐known chromophores. [ 6 ] However, this technique only partially solves the abovementioned problems in optical imaging, since it requires a high power ultrashort‐pulsed laser due to the usually low NIR absorption cross‐section of most chromophores, apart from the other issues (photostability and photobleaching, as well as a short luminescent lifetime of the luminophore).…”