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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??m and 12??m, with a field-of-view as large as 450??m. 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??m 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.

SUBMITTER: Ducourthial G

PROVIDER: S-EPMC4682136 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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

Ducourthial Guillaume G Leclerc Pierre P Mansuryan Tigran T Fabert Marc M Brevier Julien J Habert Rémi R Braud Flavie F Batrin Renaud R Vever-Bizet Christine C Bourg-Heckly Geneviève G Thiberville Luc L Druilhe Anne A Druilhe Anne A Kudlinski Alexandre A Louradour Frédéric F

Scientific reports 20151217

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 pol ...[more]

PMID: 26673905

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Project description:Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell's own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine-luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine-luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine-luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.

Dataset Information

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

Publications

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

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