In vivo three-photon imaging of subcortical structures of an intact mouse brain using quantum dots

Multiphoton microscopy is a valuable technique for producing high-resolution images deep within intact biological tissue in vivo. Furthermore, multiphoton microscopes that utilize longer-wavelength excitation at ~1700 nm as well as higher-order excitation processes permit deeper imaging within the same tissue [1]. We recently developed a laser source that produces 65 fs pulses at 1,675 nm excitation using a 1,550 nm laser (Calmar) and a photonic crystal rod (NKT photonics), which we use to perform three-photon imaging experiments [1]. In a recent publication, we showed that we are capable of producing images within the CA1 region of the hippocampus of the intact mouse brain in vivo using Texas Red-dextran (vascular label) and red fluorescent-protein (neuron label); hippocampal imaging within the intact mouse brain is practically impossible using two-photon microscopy due to the highly scattering External Capsule (EC, a.k.a. “white matter”) that separates the neocortex from the hippocampus [1]. Furthermore, the measured signal-to-background ratio (SBR), which determines the maximum imaging depth within tissue, ranged between 79 and 119 at depths between 1,000 and 1,135 μm (within the hippocampus). Therefore, the imaging depth of the three-photon microscopy demonstrated was limited by noise instead of the fundamental SBR, and a brighter fluorophore will allow deeper imaging using the same three-photon approach [1].