Polymer dots enable deep in vivo multiphoton fluorescence imaging of microvasculature.

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep MPM has never before been demonstrated. Herein, we characterize the multiphoton properties of three pdot variants and perform deep in vivo MPM imaging of cortical rodent microvasculature. We find pdot brightness exceeds conventional fluorophores, including quantum dots, and their broad multiphoton absorption spectrum permits imaging at wavelengths better-suited for biological imaging and confers compatibility with a range of longer excitation wavelengths. This results in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths (z = 1,300 µm). Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, enabling interrogation of deep structures in vivo.