Regulation of physiological pH is integral for proper whole body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, composed of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8 and 8.4, making it a viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished using both common luminescence spectroscopy and advanced optical imaging methods. Using an IVIS Spectrum, pH can be measured through tissue with Ratio-pHCL-1, which is shown in vitro and calibrated in sacrificed mouse models. Intraperitoneal injections of Ratio-pHCL-1 into live mice show high photon outputs and consistent increases in the flux ratio when measured at pH 6, 7, and 8.

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Spirolactam rhodamines are fluorescent photoswitches that are useful for single molecule localization microscopy, volumetric 3D digital light photoactivatable dye displays, and other applications. Measurement of the photophysical properties, particularly photoswitching kinetics and quantum yields, is challenging and a comprehensive understanding of how molecular structure affects these parameters remains incomplete. In this study, we have synthesized a series of N‐aryl spirolactam rhodamine photoswitches with fluorescent emissions at 585 nm and 518 nm. Extinction coefficients and fluorescence quantum yields of the fluorescent form of the photoswitch have been measured using excess trifluoroacetic acid to drive the equilibrium to the open form. A method to determine photoswitching kinetics and quantum yields was developed by monitoring the kinetics to reach equilibrium between the on‐state and off‐state and fitting this data to a rate equation for a reaction in equilibrium. Trends based on the electronic and steric properties of the aryl substituents were evaluated. Using this information, a proof‐of‐principle demonstration of 3D voxel formation was accomplished using a green (518 nm) emitting photoswitch, setting the foundation for a multi‐colour volumetric 3D display.

Chemiluminescent molecules which emit light in response to a chemical reaction are powerful tools for the detection and measurement of biological analytes and enable the understanding of complex biochemical processes in living systems. Triggerable chemiluminescent 1,2-dioxetanes have been studied and tuned over the past decades to advance quantitative measurement of biological analytes and molecular imaging in live cells and animals. A crucial determinant of success for these 1,2-dioxetane based sensors is their chemical structure, which can be manipulated to achieve desired chemical properties. In this Perspective, we survey the structural space of triggerable 1,2-dioxetane and assess how their design features affect chemiluminescence properties including quantum yield, emission wavelength, and decomposition kinetics. Based on this appraisal, we identify some structural modifications of 1,2-dioxetanes that are ripe for exploration in the context of chemiluminescent biological sensors.

The controlled introduction of defects into MOFs is a powerful strategy to induce new physiochemical properties and improve their performance for target applications. Herein, we present a new strategy for...