Characterizing the depth dependence of resolution and signal strength for two-photon microscopy

Two-photon (2P) microscopy is a useful tool for studying structure and function of biological samples. As optical optimizations occur in 2P systems that allow imaging at deeper depths, there is a need to characterize the resolution and the signal interactions with tissue at these depths. Here, we discuss processes to determine the resolution of a 2P microscope using sub-resolution sized micro-spheres to mimic point spread functions. Through this process, the resolution of the microscope was determined to be about 0.942 μm in vitro and about 1.08 μm in vivo, values that did not change with respect to depth. Additionally, we investigated the relationship between contrast, background intensity, and noise with depth in vivo. From this study, contrast decreased with depth, while background intensity and noise both increased. These results suggest that the decrease in resolving power at deep depths is likely due to the inability to differentiate signal from background and not due to a decrease in the overall resolution of the system.