Multimodal Multiphoton Tomography with a Compact Femtosecond Fiber Laser

Abstract

Multiphoton Tomography (MPT) based on near infrared femtosecond laser technology has become a versatile high resolution clinical and research imaging tool. We report on multimodal MPT with the air-cooled fiber-laser tomograph MPTcompact. The ultracompact passively mode locked Erbium doped 50/80 MHz laser operating at 780 nm is directly integrated into the 360° imaging head mounted on a flexible mechanical arm. The tomograph provides optical biopsies with subcellular resolution and optical metabolic imaging (OMI) capability based on two-photon autofluorescence (AF), second harmonic generation (SHG), fluorescence lifetime imaging (FLIM) by time-correlated single photon counting with 250 ps temporal resolution, reflectance confocal microscopy (RCM), and white LED light CMOS camera imaging. For the first time, near infrared femtosecond laser pulses have been used on human skin to realize simultaneous one-photon confocal imaging and two-photon imaging. Most useful information is provided by the two-photon excited intratissue autofluorescence of the coenzymes NAD(P)H and flavins. The signals of both types of coenzymes can be separated by FLIM. Furthermore, the free and protein-bound form can be distinguished by time-resolved autofluorescence detection because the protein-bound NADH has one order higher autofluorescence lifetime than free (non-bound) NADH. The tomograph contains onboard storage batteries so that it can operate for up to a few hours independently form external power supply. Applications include cancer detection and in situ evaluation of anti-ageing drugs and pharmaceuticals. The tomograph MPTcompact has been successfully tested in a clinical multicenter study for the diagnosis of malignant melanoma on 100 patients with suspicious pigmented lesions. Multiphoton tomography has the potential to realize non-invasive high-resolution label-free in vivo histology within minutes and, therefore, to reduce the number of physically taken biopsies.