Rigid and high-numerical-aperture two-photon fluorescence endoscope.

We present a rigid miniaturized optical system block fiber-optic two-photon endoscope based on a compact two-axis piezo scanner system and a miniature high (0.65) NA GRIN lens objective. The optical system is scanned as a whole by a piezo scanner allowing always an on-axis beam irradiation of the optical system. A photonic crystal fiber is used for excitation and ultrashort laser pulses can be delivered with typical power up to 100 mW at 800 nm. Two-photon fluorescence signal is collected by the use of a multimode fiber. Lateral resolution values for the system were experimentally measured to be 0.67 microm vertically and 1.08 microm horizontally. Axial resolution was found to be 5.8 microm. The endoscope is highly flexible and controllable in terms of time acquisition, resolution, and magnification. Fluorescence images were acquired over a 420 microm x 420 microm field of view. Results presented here demonstrate the ability of the system to resolve subcellular details and the potential of the technology for in vivo applications.