Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal.

Guillaume Ducourthial,Julien Brevier,Luc Thiberville,Alexandre Kudlinski,Pierre Leclerc,Tigran Mansuryan,Frédéric Louradour,Geneviève Bourg-Heckly,Marc Fabert,Renaud Batrin,Flavie Braud,Rémi Habert,Christine Vever-Bizet,Anne Druilhe

doi:10.1038/srep18303

Abstract

We present a two-photon microendoscope capable of in vivo label-free deep-tissue high-resolution fast imaging through a very long optical fiber. First, an advanced light-pulse spectro-temporal shaping device optimally precompensates for linear and nonlinear distortions occurring during propagation within the endoscopic fiber. This enables the delivery of sub-40-fs duration infrared excitation pulses at the output of 5 meters of fiber. Second, the endoscopic fiber is a custom-made double-clad polarization-maintaining photonic crystal fiber specifically designed to optimize the imaging resolution and the intrinsic luminescence backward collection. Third, a miniaturized fiber-scanner of 2.2 mm outer diameter allows simultaneous second harmonic generation (SHG) and two-photon excited autofluorescence (TPEF) imaging at 8 frames per second. This microendoscope’s transverse and axial resolutions amount respectively to 0.8 μm and 12 μm, with a field-of-view as large as 450 μm. This microendoscope’s unprecedented capabilities are validated during label-free imaging, ex vivo on various fixed human tissue samples, and in vivo on an anesthetized mouse kidney demonstrating an imaging penetration depth greater than 300 μm below the surface of the organ. The results reported in this manuscript confirm that nonlinear microendoscopy can become a valuable clinical tool for real-time in situ assessment of pathological states.

Highlights

The microscopy probe through its wide-field imaging channel
We present an advanced two-photon microendoscope (TPME) involving a custom-made air-silica double-clad photonic crystal fiber (DC-PCF), which is associated to an efficient fiber dispersion and nonlinearity pre-compensation device
We reported a multimodal TPME fulfilling most of the requirements of clinical endoscopy, while presenting performances comparable to those of a bench-top two-photon microscope

Summary

Introduction

The microscopy probe through its wide-field imaging channel. other important characteristics of the ideal TPME rely on its field of view (FOV) and working distance (WD). We present an advanced TPME involving a custom-made air-silica double-clad photonic crystal fiber (DC-PCF), which is associated to an efficient fiber dispersion and nonlinearity pre-compensation device. This strategy enables the delivery of energetic sub-40 fs IR pulses at the output of a 5-m-long DC-PCF with a 3.5 μ m diameter inner core. Efficient backward VIS collection and guidance is obtained by means of a large area high numerical aperture concentric cladding which acts as a second collecting core around the photonic crystal microstructure These key features allow the demonstration of a fully operating TPME system whose distal part is embedded inside a 2.2 mm outer diameter probe. We demonstrate that our miniature fiber-based system has performances approaching those of its table-top counterparts while involving a very long and flexible optical fiber, successfully meeting most of the requirements for future clinical imaging

Methods

Results

Conclusion