Development of a versatile two-photon endoscope for biological imaging

We describe a versatile, catheter-type two-photon probe, designed for in vivo and ex vivo imaging of the aqueous outflow pathway in the eye. The device consists of a silica double cladding fiber used for laser delivery and fluorescence collection, a spiral fiber scanner driven by a miniature piezoelectric tube, and an assembly of three micro-size doublet achromatic lenses used for focusing the laser and collecting the two-photon excitation signal. All the components have a maximum diameter of 2 mm and are enclosed in a length of 12-gauge stainless steel hypodermic tubing having an outer diameter of 2.8 mm. The lateral and axial resolutions of the probe are measured to be 1.5 μm and 9.2 μm, respectively. Different lens configurations and fibers are evaluated by comparing their spatial resolutions and fluorescence signal collection efficiencies. Doublet achromatic lenses and a double cladding fiber with a high inner cladding numerical aperture are found to produce a high signal collection efficiency, which is essential for imaging live tissues. Simple methods for reducing image distortions are demonstrated. Images of human trabecular meshwork tissue are successfully obtained with this miniature two-photon microscope.