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Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy.

ABSTRACT: Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO₄ and BaTiO₃ nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow.

SUBMITTER: Malkinson G

PROVIDER: S-EPMC7735018 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Publications

Fast <i>In Vivo</i> Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy.

Malkinson Guy G Mahou Pierre P Chaudan Élodie É Gacoin Thierry T Sonay Ali Y AY Pantazis Periklis P Beaurepaire Emmanuel E Supatto Willy W

ACS photonics 20200228 4

Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structu ...[more]

PMID: 33335947

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Project description:Live observation of biological phenomena in the context of living organisms can provide important insights in the mechanisms of these phenomena. However, the spatially complex and dynamic physiology of multicellular organisms can be a challenging environment to make observations with fluorescence microscopy. Due to the illumination of out-of-focus planes, confocal and particularly widefield fluorescence microscopy suffer from low signal-to-background ratio (SBR), photo toxicity and bleaching of fluorescent probes. In light-sheet microscopy (LSM), solely the focal plane of the detection objective is illuminated, minimising out-of-focus fluorescence and photobleaching, thereby enhancing SBR, allowing for low laser intensities and longer acquisition periods. Here we present a straightforward light-sheet microscope with a 1.0-NA detection objective and a fast sample-positioning stage that allows for four degrees of freedom. By imaging the sensory cilia and nervous system of living young adult C. elegans, we demonstrate that the instrument is well suited for relatively fast, volumetric imaging of larger (hundreds of micrometres cubed) living samples. These experiments demonstrate that such an instrument provides a valuable addition to commonly used widefield and confocal fluorescence microscopes. LAY DESCRIPTION: In fluorescence microscopy, sharp images can only be obtained when the light obtained from the section of the image that is in focus is not overwhelmed by light emerging from elsewhere. In this paper, we present a light-sheet fluorescence microscope, based on the OpenSPIM initiative, with a magnification of 90× and a sensitive sample positioning stage that allows for fast controlled linear movement and rotation. In a light-sheet microscope (LSM), the sample is illuminated from the side, compared to the direction of detection, limiting illumination only to the part of the sample that is imaged in the focal plane (general resources: Wikipedia or MicroscopyU). This does not only limit background noise, but also reduces damage to the sample due to phototoxicity. This makes a LSM particularly suitable for imaging living samples at high resolution, in three dimensions, over long periods of time. Our instrument was specifically designed for imaging adult C. elegans nematodes. We show here how the instrument compares to a standard epifluorescence microscope, imaging neuronal structures in the animals. The instrument proved well suited for fast volumetric imaging of larger cellular structures such as C. elegans neuronal cell bodies. Our experiments show that the instrument provides a valuable addition to widefield and confocal fluorescence microscopes commonly used to image adult C. elegans.

Dataset Information

Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy.

Publications

Fast <i>In Vivo</i> Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy.

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