Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal
Guillaume DucourthialPierre LeclercTigran MansuryanMarc FabertJulien BrévierRémi HabertFlavie BraudRenaud BatrinChristine Vever‐BizetGeneviève Bourg-HecklyLuc ThibervilleAnne DruilheAlexandre KudlinskiFrédéric Louradour
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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.Keywords:
Two-photon excitation microscopy
Endomicroscopy
In this paper, we demonstrate that the penetration depth under two-photon excitation is limited by the strength of two-photon fluorescence and is not necessarily larger than that under single-photon excitation, although image resolution is much higher in the former case.
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Fiber-optic-based two-photon fluorescence endomicroscopy is emerging as an enabling technology for in vivo histological imaging of internal organs and functional neuronal imaging on freely-behaving animals. However, high-speed imaging remains challenging due to the expense of miniaturization and lack of suited fast beam scanners. For many applications, a higher imaging speed is highly desired, especially for monitoring functional dynamics such as transient dendritic responses in neuroscience. This Letter reports the development of a fast fiber-optic scanning endo-microscope with an imaging speed higher than 26 frames/s. In vivo neural dynamics imaging with the high-speed endomicroscope was performed on a freely-behaving mouse over the primary motor cortex that expressed GCaMP6m. The results demonstrate its capability of real-time monitoring of transient neuronal dynamics with very fine temporal resolution.
Endomicroscopy
Fluorescence-lifetime imaging microscopy
Temporal resolution
Two-photon excitation microscopy
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Our contribution is focused on broadening of the spectrum of available non-linear optical (NLO)-phores (contrast agents for nonlinear optical microscopy) by design and synthesis of new organic dyes with appropriate optical properties. One of the main pre-requisites of microscopy utilizing non-linear excitation is the existence of molecules that are able to provide NLO response for the second-harmonic generation (SHG) or for the two-photon excited fluorescence (TPEF). Many molecules naturally occurring in living tissue such as collagens or NAD(P)H were successfully used in this regard, but there is a natural interest in broadening of the spectrum of available NLO-phores. Gathered results confirm applicability of the newly synthesized dyes as new potential NLO-phores for confocal laser scanning microscopy with nonlinear excitation in rat aorta.
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Second-harmonic imaging microscopy
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We demonstrate in vivo microvasculature and blood flow imaging based on two-photon excited autofluorescence of blood plasma and NADH in zebrafish. The blood flow images enable counting circulating leukocyte for detecting acute sterile inflammation.
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Two-photon excitation microscopy
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Real-time in vivo microscopic imaging has become a reality with the advent of confocal and nonlinear endomicroscopy. These devices are best utilized in conjunction with standard white light endoscopy. We evaluated the use of fluorescence endomicroscopy in detecting microscopic abnormalities in colonic tissues. Mice of C57bl/6 strain had intraperitoneal injection with azoxymethane once every week for five weeks and littermates, not exposed to azoxymethane served as controls. After 14 weeks, intestines were imaged by fluorescence endomicroscopy. The images show obvious cellular structural differences between those two groups of mice. The difference in endomicroscopy imaging can be used for identifying tissues suspicious for neoplasia or other changes, leading to early diagnosis of gastrointestinal track of cancer.
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OCT tethered capsule endomicroscopy (TCE) is an emerging noninvasive diagnostic imaging technology for gastrointestinal (GI) tract disorders. OCT measures tissue reflectivity that provides morphologic image contrast, and thus is incapable of ascertaining molecular information that can be useful for improving diagnostic accuracy. Here, we introduce an extension to OCT TCE that includes a fluorescence (FL) imaging channel for attaining complementary, co-registered molecular contrast. We present the development of an OCT-FL TCE capsule and a portable, plug-and-play OCT-FL imaging system. The technology is validated in phantom experiments and feasibility is demonstrated in a methylene blue (MB)-stained swine esophageal injury model, ex vivo and in vivo .
Endomicroscopy
Fluorescence-lifetime imaging microscopy
Molecular Imaging
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A malignant tumor remains one of the leading causes of deaths across the world. Thus, diagnosis of tumor development with noninvasive visualizing methods is significant for tumor therapy. Herein, an activatable two-photon NIR fluorescent probe DHQ-Rd-PN for in vivo imaging of peroxynitrite in a tumor was elaborately designed. The probe demonstrated an increased NIR emission in response to peroxynitrite in vitro, which ensured that the probe detects ONOO– in cell and in vivo. Cellular imaging results disclosed that the probe was competent to detect adscititious ONOO– level change in HeLa cells, as well as endogenous ONOO– concentration in lipopolysaccharides (LPS) and IFN-γ-stimulated RAW 264.7 cells. Additionally, zebrafish in vivo imaging revealed that the probe accumulated in the pancreas and was lightened up by the addition of ONOO–. Remarkably, the probe can be harnessed to image an ONOO– production profile in xenograft 4T1 tumor mice by both one-photon and two-photon in vivo fluorescence imaging. Benefiting with the two-photon excitable properties and NIR emissive properties, the probe can be used for noninvasive in vivo imaging of ONOO– in the onset and development of tumors for the first time. This work provided a noninvasive and efficient detection method for ONOO– in a tumor, which would find more applications in tumor diagnosis and therapies.
Two-photon excitation microscopy
HeLa
Fluorescence-lifetime imaging microscopy
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A compact endomicroscope is the only solution for transferring second harmonic generation (SHG) imaging into in vivo imaging and real time monitoring the content and structure of collagen. This is important for early diagnoses of different diseases associated with collagen change. A compact nonlinear endomicroscope using a double clad fiber (DCF) is newly employed in SHG imaging. The experiment shows the core of the DCF can maintain the linear polarization of the excitation laser beam in particular directions, and the degree of polarization of the excitation laser beam directly affects signal to noise ratio of SHG imaging. The nonlinear endomicroscope can display clear three dimensional (3D) SHG images of mouse tail tendon without the aid of contrast agents, which reveals the collagen fiber structure at different depths. The high resolution of SHG imaging from the endomicroscope shows that SHG imaging can reveal additional information about the orientation and degree of organisation of proteins and collagen fibers than two-photon-excited fluorescence imaging. Therefore SHG imaging offers endomicroscopy with additional channel of imaging for understanding more about biological phenomena.
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Endomicroscopy
Fluorescence-lifetime imaging microscopy
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Deep tissue in vivo two-photon fluorescence imaging of cortical vasculature in a mouse brain using 1280-nm excitation is presented. A record imaging depth of 1.6 mm in mouse cortex is achieved in vivo, approximately reaching the fundamental depth limit in scattering tissue.
Two-photon excitation microscopy
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