After euthanizing mice, the NALT and lymph nodes were surgically isolated and transferred to an 25x25x1 mm imaging chamber (Grace Bio-Labs, Bend, OR) containing RPMI. Two-photon imaging was performed using an Olympus Fluoview FV1200MPE microscope with a 40X/0.8NA water emersion objective lens. For multiphoton excitation and second harmonic generation, a MaiTai Ti:sapphire laser (Newport/Spectra-Physics, Santa Clara, CA) was tuned to 790 and 890 nm for optimized excitation of mCherry expressing Salmonella and CD11c-eYFP+ cells, respectively. Emission filters were 420–460 for SHG, 495–540 nm for eYFP, and 575–630 nm for mCherry. Six-eight 80 μm z-stacks were acquired for each NALT and two-four 200 μm z-stacks were acquired for each lymph node with 2 μm z-spacing between each optical section (512x512 pixels). 3-D reconstruction and image analysis was performed using watershed segmentation algorithms in Imaris (Bitplane, South Windsor, CT) to quantify numbers of mCherry+ and CD11c-eYFP+ cells.
Fluoview fv1200mpe microscope
Manufactured by Olympus
The Fluoview FV1200MPE microscope is a multi-photon excitation laser scanning microscope designed for high-resolution imaging of biological samples. It provides high-sensitivity detection and fast image acquisition capabilities.
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2 protocols using fluoview fv1200mpe microscope
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Imaging Immune Cell Dynamics in NALT
2
Imaging Immune Cell Dynamics in NALT
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After euthanizing mice, the NALT and lymph nodes were surgically isolated and transferred to an 25x25x1 mm imaging chamber (Grace Bio-Labs, Bend, OR) containing RPMI. Two-photon imaging was performed using an Olympus Fluoview FV1200MPE microscope with a 40X/0.8NA water emersion objective lens. For multiphoton excitation and second harmonic generation, a MaiTai Ti:sapphire laser (Newport/Spectra-Physics, Santa Clara, CA) was tuned to 790 and 890 nm for optimized excitation of mCherry expressing Salmonella and CD11c-eYFP+ cells, respectively. Emission filters were 420–460 for SHG, 495–540 nm for eYFP, and 575–630 nm for mCherry. Six-eight 80 μm z-stacks were acquired for each NALT and two-four 200 μm z-stacks were acquired for each lymph node with 2 μm z-spacing between each optical section (512x512 pixels). 3-D reconstruction and image analysis was performed using watershed segmentation algorithms in Imaris (Bitplane, South Windsor, CT) to quantify numbers of mCherry+ and CD11c-eYFP+ cells.
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