Multimodal two-photon and three-photon endomicroscopy for 3D tissue imaging

Multimodal multiphoton microscopy (MPM) can provide fast, label-free, non-invasive examination of cells, extracellular matrix, and lipids. Two-photon microscopy (2PM) can detect second harmonic generation (SHG) from fibrillar collagen and striated muscle myosin, whereas two-photon excitation fluorescence (2PEF) can detect intrinsic fluorophores such as NADH from cells. Meanwhile, three-photon microscopy (3PM) can detect third harmonic generation (THG) from lipids and tissue interfaces. We have developed a miniaturized multimodal multiphoton system which can perform label-free two-photon and three-photon imaging. An Er-doped fiber laser delivers fundamental pulses at 1580 nm and 80 fs for exciting THG. SHG and 2PEF are excited at 790 nm via the frequency doubling of 1580 nm pulses. For clinical applications, a compact probe is being developed with single-mode fiber for delivering the femtosecond excitation pulses and multi-mode fiber for collecting the MPM signals. A MEMS mirror performs lateral scanning at up to 4 frames/s. For objective lenses, a miniature aspherical lens (NA=0.64) is compared with a gradient index microobjective (NA=0.8). Shape memory alloy actuator used in smartphone cameras is evaluated for shifting the focal plane to acquire Z-stacks for 3D tissue imaging. High-resolution SHG, 2PEF, and THG images are acquired from biological tissues and show that multimodal MPM endomicroscopy has great potential for clinical applications as an alternative to histology.