2.03] Development of a two-photon endoscope

Abstract

Objective An endoscopic probe is proposed for in-vivo high-resolution, two-photon imaging. This probe, based on a flexible fiber bundle combined with a miniaturized high numerical aperture objective, allows 3D-imaging of superficial tissue and therefore may be an important tool for optical biopsy. The optical sectioning strength was characterized by the axial resolution. The two-photon microendoscope may offer a favorable imaging depth within scattering tissue compared to the confocal approach. However, a disadvantage of the two-photon endoscopic application is the lower fluorescence signal due to linear and non-linear pulse distortion. Simulations and experiments were carried out to study these effects. Material and methods Two femtosecond laser sources, a Ti:sapphire laser system (Coherent, 80 MHz, 800 nm, 120 fs) and a fiber-based system (Toptica, 90 MHz, 1090 nm, 20 fs) were used in combination with various fiber bundles (Sumitomo Electrics Industries). Two miniaturized objectives were examined, a GRIN-lens system (GRINTECH) and a custom made system (Storz), with a numerical aperture of 0.75 and 0.6, respectively. The fluorescent dyes, fluorescein and cresyl violet acetate, were used on sample materials and excised bladder wall tissue. Simulations of short pulse propagation in the fiber materials were done with LAB2-A. Results First measurements with our two-photon microendoscope showed that it is possible to detect fluorescence signals without compensating for linear and non-linear pulse broadening. The outer diameter is already compatible with the working channel of a conventional cystoscope. Furthermore, the optical performance showed a good image quality at high axial resolution. Consequently, intracellular details could be resolved from bulky tissue with high-speed performance. For future optimizations, the simulations indicate an effective decrease of linear and nonlinear pulse distortions by mainly quadratic pre-chirping, which may be implemented for example, using a simple grating compressor. Conclusion The current microendoscope for two-photon imaging shows the potential to resolve intracellular details and can be integrated into a commercial endoscope. Two-photon endoscopy may be the possible next step to effectively overcome the limitations of confocal microendoscopy. In particular, the higher penetration depth may contribute to a broader acceptance for in-vivo tissue diagnostics.