Improved Sensitivity in Large Field of View Multispectral Laser‐Scanning Photoacoustic Microscopy for Measuring Oxygen Saturation In Vivo

Multispectral photoacoustic microscopy (PAM) using stimulated Raman scattering (SRS) has been employed to measure oxygen saturation (sO 2 ) in biological tissue. However, laser‐scanning photoacoustic microscopy (LS‐PAM) inherently suffers from low detection sensitivity due to the use of a flat transducer and non‐coaxial alignment of the transducer with the optical scan. Although wide‐field‐of‐view LS‐PAM has been implemented, it typically results in coarser lateral resolution and hence lower sensitivity than existing LS‐PAM systems. Here, we present a wide‐field multispectral LS‐PAM system for measuring sO 2 in biological tissue. Instead of relying on two discrete wavelengths, our method employs two wavelength groups—a isosbestic group (532 nm and 545 nm) and a deoxyhemoglobin‐dominant group (545 nm and 558 nm). We demonstrate that using these groups improves the signal‐to‐noise ratio (SNR) of the detected signals, leading to more accurate sO 2 measurements. The performance of this system is validated through both phantom and in vivo studies.