Extending the depth limit of multiphoton microscopy for in vivo brain imaging

The benefit of high-resolution imaging provided by optical microscopy has resulted in many discoveries in both biology and neuroscience. Two-photon fluorescence microscopy (2PM) is widely used for in vivo brain imaging to visualize cerebral vasculature and neuronal physiology. Conventional 2PM using titanium-doped sapphire oscillators is typically limited to imaging depths less than 600 um due to their short excitation wavelengths (700 -1,000 nm) and low pulse energy (~10 nJ). The ideal approach for deep imaging is to use both longer wavelengths to reduce the effects of scattering by heterogeneous brain tissue and higher energy pulses such that more photons reach the excitation volume at deeper tissue depths. I perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier (OPA). The OPA outputs 400 nJ pulse energies and is tunable from 1,100 to 1,400 nm. The tunability of the OPA is an advantage over other high-pulse-energy lasers because the OPA wavelength can be matched to the peak absorption of the target fluorophore, enabling the excitation of numerous different fluorophores. I demonstrate an imaging depth of 1,200 um in vasculature labeled with Texas Red and 1,160 um in neurons labeled with tdTomato, and perform line scans as deep as 1200 um to measure the blood flow speed in a single capillary. I also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.