Single-wavelength excited photoacoustic-fluorescence microscopy for in vivo pH mapping

The balance between radiative and non-radiative decay is affected by various factors at the excitation state. A simultaneous acquisition of the fluorescence and photoacoustic signals from an appropriate probe provides an efficient, high-resolution means to monitor such a balance in a biological target, and thus may render its physiological information. Acidity plays an important role in tissue physiology. Here, we report an integrated photoacoustic-fluorescence microscopy (PA-FLM) for high-resolution (<3  μm) image mapping of interstitial pH by detecting the shift in the signal balance of a pH-sensitive probe. The hypothesis and the technical feasibility are validated with an in vivo tumor model. The results show that the technique can effectively monitor pH changes within the range of biological acidity and are independent of the excitation source fluctuation and local probe concentration. We thus propose that, with further research and selection of proper probes, PA-FLM may provide a potential alternative for monitoring tissue physiology.