Label-free multimodal imaging with simultaneous two-photon and three-photon microscopy and kernel-based nonlinear scaling denoising.

We report on a compact multimodal imaging system that can acquire two-photon microscopy (2PM) and three-photon microscopy (3PM) images simultaneously. With dual excitation wavelengths, multiple contrasts including two-photon-excitation-fluorescence (2PEF), second harmonic generation (SHG), and third harmonic generation (THG) are acquired simultaneously from cells, collagen fibers, and interfaces, all label-free. Challenges related to the excitation by two wavelengths and the effective separation of 2PM and 3PM signals are discussed and addressed. The data processing challenge where multiple contrasts can have significantly varying signal levels is also addressed. A kernel-based nonlinear scaling (KNS) denoising method is introduced to reduce noise from ultra-low signal images and generate high-quality multimodal images. Simultaneous 2PM and 3PM imaging is demonstrated on various tissue samples. The simultaneous acquisition speeds up the imaging process and minimizes the commonly encountered problem of motion artifacts and mechanical drift in sequential acquisition. Multimodal imaging with simultaneous 2PM and 3PM will have great potential for label-free