Background-Free Referenced Luminescence Sensing and Imaging of pH Using Upconverting Phosphors and Color Camera Read-out

Fluorescence background and problems with proper signal referencing severely disrupt the read-out of luminescence sensors and images. We present a pH sensor film in combination with a simple read-out technique that overcomes issues of background signals and autofluorescence. It consists of micrometer-sized upconversion phosphors (UCPs) and a pH indicator (Neutral Red) that absorbs their green emission. Both are embedded in a proton permeable hydrogel matrix. The UCPs generate green and red luminescence upon excitation with IR light of 980 nm wavelength. Solely the green light of the UCPs is affected by the pH indicator, while the red emission acts as inert reference signal for ratiometric measurements. The emission peaks of the UCPs match the red and green color channels of standard digital cameras. Thereby, the devised sensor film can be used for referenced ratiometric sensing and 2D imaging of pH using a color camera read-out. The sensor setup using common and hand-held devices is cheap and straightforward and allows for point-of-care measurements. Finally, pH measurements in human serum samples show the potential of this sensor for imaging free of interfering background signals

Environmental Impact on the Excitation Path of the Red Upconversion Emission of Nanocrystalline NaYF₄:Yb³⁺,Er³⁺

The mechanism for red upconversion luminescence of Yb–Er codoped materials is not generally agreed on in the literature. Both two-photon and three-photon processes have been suggested as the main path for red upconversion emission. We have studied β-NaYF₄:Yb³⁺,Er³⁺ nanoparticles in H₂O and D₂O, and we propose that the nanoparticle environment is a major factor in the selection of the preferred red upconversion excitation pathway. In H₂O, efficient multiphonon relaxation (MPR) promotes the two-photon mechanism through green emitting states, while, in D₂O, MPR is less effective and the three-photon path involving back energy transfer to Yb³⁺ is the dominant mechanism. For the green upconversion emission, our results suggest the common two-photon path through the ⁴F_9/2 energy state in both H₂O and D₂O

Ratiometric Sensing and Imaging of Intracellular pH Using Polyethylenimine-Coated Photon Upconversion Nanoprobes

Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in <i>in vitro</i> models with high biological background fluorescence and in <i>in vivo</i> applications, e.g., for the bioimaging of small animal models