Optimized Fluorescent pH-Sensitive Dextran Conjugates Enable Ratiometric Measurement of Cerebral Blood pH In Vivo and In Situ

Monitoring transient pH variations in vivo remains challenging, particularly in blood, a highly absorbing and dynamic tissue where sustained probe circulation is required. To address this, we developed a dual-emission ratiometric pH probe approach based on dextran conjugates to ensure stability in the circulation comprising (1) AF488, a pH-insensitive fluorophore, and (2) H-Ruby (HR), a red-emitting, fluorogenic, pH-sensitive dye. While H-Rubies are efficient pH indicators, their conjugation to dextran induces strong aggregation-caused quenching (ACQ), which limits both their loading capacity and brightness. To overcome this, we designed a PEGylated version of H-Ruby (HR-PEG), which displays improved water solubility and two-photon absorption while retaining a pKa of 7.5, suitable for physiological measurements. Upon conjugation to dextran, HR-PEG exhibits significantly reduced ACQ, resulting in brighter conjugates with preserved pKa and a 12-fold fluorescence increase in response to acidification from pH 7.4 to pH 6.4. These enhancements improved signal detection in blood vessels with in vivo two-photon imaging in mice and significantly increased fluorescence responses to blood acidification, thereby allowing accurate and quantitative measurement of the vascular acidosis caused by a brief hypercapnia.