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

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.

Second‐harmonic imaging microscopy for identifying colorectal intraepithelial neoplasia

“…All of this information can be used to assess whether a tissue is normal, LGIN or HGIN. Moreover, SHG microscopy does not require staining and in the future could be applied in vivo in a clinical setting with multiphoton endoscopy (Fu et al, 2006;Le Harzic et al, 2008;Brown et al, 2012;Ducourthial et al, 2015). With continued improvement and refinement of this technique, we believe that second-harmonic imaging microscopy will be an efficient surveillance tool for colorectal intraepithelial neoplasia and lead to better management of these lesions.…”

In vitro methods to study bubble-cell interactions: Fundamentals and therapeutic applications

“…With such tools, the imaging depth can be increased to beyond 1 mm. 187 Alternatively, thicker samples can be imaged with a significant improvement in depth by twophoton or multiphoton microscopy 188,189 or prepared post-experiment to offer better imaging capabilities off-line (see Sec. IV D).…”

New frontiers in intravital microscopy of the kidney

“…There is currently much interest in the potential usage of such instruments to perform 'virtual biopsies' in humans, whereby the histological nature of suspicious lesions could be assessed in situ [18]. It has recently been demonstrated that endoscopic microscopy can also be used to image the kidney in living anaesthetized mice [19], and although the quality of images that can be acquired is not as high as conventional MPM, this approach could clearly be advantageous for repetitive imaging studies in the same animal, perhaps even when awake.…”