Chromatic two-photon fluorescence microsopy using band-shifting imaging probes

Chromatic two-photon fluorescence microscopy using band-shifting imaging probes Two-photon fluorescence microscopy has emerged as one of the most important imaging modalities for biological research and medical diagnosis. Although many techniques exist for realizing high speed two-photon imaging in the lateral directions, the axial imaging speed is still often limited by the slow mechanical scanning of the objective lens or the specimen, presenting a significant challenge for monitoring fast biological processes at multiple depths as well as in 3D and the development of miniature endoscopy. To overcome this limitation, here a new spectrally encoded two-photon imaging technique is proposed using band-shifting imaging probes, which can enable parallel axial imaging. Specifically, different excitation wavelengths are focused onto different axial positions (through purposely introduced chromatic aberration) to excite two-photon fluorescence from the band-shifting imaging probes, which shift the emission band when the excitation wavelength varies. As such, the fluorescence signals at different axial positions are spectrally encoded to exhibit different spectral bands, and can thus be imaged in parallel by using a spectrometer or arrayed wavelength-resolving detectors. The proposed band-shifting imaging probes will be synthesized, optimized, and used for cellular labeling. Systematic characterization on the molecular structures, molecular weight, photophysical and two-photon properties will be performed. The proposed chromatic two-photon imaging system will be designed, developed, and optimized. System metrics including the mapping relationship between the axial position and fluorescence band shift, the axial imaging range, and spatial resolutions will be characterized. The proposed method will be demonstrated and validated by performing imaging of cells and tissue phantoms as well as by in vivo imaging studies of a colorectal cancer xenograft model.