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Ratiometric sensing and imaging of intracellular pH using polyethyleneimine-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 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, that 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 in vitro models with high biological background fluorescence and in in vivo applications, e.g. for the bioimaging of small animal models.