Optimal Generation of Ten Individual Green-to-Red Raman Source for Wavelength-Dependent Real-Time OR-PAM Images

In this study, ten individual green-to-red stimulated Raman scattering (SRS) peaks are optimally generated for wavelength-dependent real-time optical-resolution photoacoustic microscopy (OR-PAM) with contrast agents. Exogenous contrast agents, such as dyes, semiconducting polymers, and metallic nanoparticles, are frequently used in OR-PAM to provide structural, functional, and molecular images of biological samples with a high spatial resolution. For an effective use of various exogenous contrast agents for functional or molecular OR-PAM, selectable visible and near-infrared wavelengths are needed to select the absorption wavelengths of the contrast agents. Therefore, we optimized an SRS source, which can selectively generate ten narrow-band SRS peaks with a bandwidth smaller than 11 nm and individually produce sufficient threshold pulse energies (116–756 nJ) at a high pulse repetition rate (PRR) of 300 kHz. The wavelength-dependent photoacoustic signal was easily obtained in a methylene blue phantom and in vivo mouse studies using the optimal generation conditions of the ten-wavelength green-to-red SRS source, from 545 to 695 nm. In particular, for the first time, wavelength-dependent real-time in vivo OR-PAM images at a PRR of hundreds of kilohertz were obtained to distinguish gold nanoparticles from distributed blood vessels of a mouse ear.