Spinning disk confocal intravital microscopy was performed using an Olympus BX51WI (Olympus, Center Valley, PA) upright microscope equipped with a 20×/0.95 XLUM Plan Fl water immersion objective. The microscope was equipped with a confocal light path (WaveFx, Quorum, Guelph, ON) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation, Tokyo, Japan). Laser excitation at 488, 561 and 649nm (Cobalt, Stockholm, Sweden), was used in rapid succession and fluorescence in green, red and blue channels was visualized with the appropriate long pass filters (Semrock, Rochester, NY). Exposure time for all wavelengths was between 500 and 600ms. Sensitivity settings were maintained at the same level for all experiments. A 512×512 pixels back-thinned EMCCD camera (C9100–13, Hamamatsu, Bridgewater, NJ) was used for fluorescence detection. Volocity Acquisition software (Improvision Inc., Lexington, MA) was used to drive the confocal microscope. Images captured using the spinning disk were processed and analyzed in Volocity 6.0.1. NET area and NET number were quantified using the Volocity software.
Csu 10 head
Manufactured by Yokogawa
Sourced in Canada
The CSU-10 is a compact spinning-disk confocal scanner unit designed for high-speed confocal imaging. It features a high-speed confocal scanner with a high-intensity white light LED illumination source and a sensitive scientific CMOS camera. The CSU-10 is capable of capturing high-quality confocal images at fast frame rates.
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Lab products found in correlation
Wavefx, by Quorum Technologies (4 mentions)
Long pass filter, by IDEX Corporation (2 mentions)
Wavefx, by Yokogawa (2 mentions)
Ix81 inverted microscope, by Olympus (2 mentions)
Volocity software, by PerkinElmer (2 mentions)
Bx51wi, by Olympus (1 mentions)
C9100 13, by Hamamatsu Photonics (1 mentions)
Anti pd l1 pe clone 10f 9g2, by BioLegend (1 mentions)
Anti pd 1 apc clone 29f 1a12, by BioLegend (1 mentions)
Volocity, by Quorum Technologies (1 mentions)
7 protocols using csu 10 head
1
Intravital Spinning Disk Confocal Microscopy
2
In Vivo Imaging of Liver Immune Cells
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A tail vein catheter was inserted into mice after anesthetization with 200 mg/kg ketamine and 10 mg/kg xylazine. Mouse body temperature was maintained at 37 °C with a heated stage. Image acquisition was performed using an Olympus IX81 inverted microscope, equipped with an Olympus focus drive and a motorized stage, and coupled to a confocal light path based on a modified Yokogawa CSU-10 head. Target cells were visualized using fluorescently stained antibodies. KCs and B cells were stained by i.v. injection of 2.5 μg anti–F4-80 (clone BM8) or 3.5 μg anti-CD19 (clone 1D3) fluorescent conjugated mAbs. Volocity v7 and Image J v1.44 software were used to drive the confocal microscope, for 3D rendering, acquisition, and analysis of images, as well as to track iNKT accumulation and interactions with KCs and B cells. We used 2 mm2 stitch images of the liver making use of the find object function and appropriate thresholding72 (link).
3
In Vivo Microscopy of Platelet Dynamics
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Mice were anesthetized using 10 mg/kg xylazine hydrochloride and 200 mg/kg ketamine hydrochloride, and livers, spleens, and lungs were prepared for IVM as previously described (Surewaard and Kubes, 2017 (link); Deniset et al., 2017 (link); Thanabalasuriar et al., 2017 (link)). A jugular vein catheter was inserted to inject fluorescent antibodies and to maintain anesthesia. Image acquisition (liver and spleen) was performed using an inverted microscope (Olympus IX81) equipped with a focus drive (Olympus) and motorized stage (Applied Scientific Instrumentation) and fitted with a motorized objective turret equipped with 4×/0.16 UPLANSAPO, 10×/0.40 UPLANSAPO, and 20×/0.70 UPLANSAPO objective lenses. Image acquisition (lung) was performed with an upright microscope (BX51; Olympus) using a ×20/0.95W NA water XLUM Plan F1 objective. The microscopes were equipped with a confocal light path (Quorum Technologies WaveFx) based on a modified CSU-10 head (Yokogawa Electric Corporation). Kupffer cells, platelets, and the endothelium were visualized using fluorescently labeled antibodies. For each animal, platelet accumulation was recorded in three randomly selected fields of view. Sialidase was injected i.v. 2 min after image acquisition was started. Volocity software (PerkinElmer) was used to quantify platelet accumulation over time.
4
Spinning Disk Confocal Microscopy of Mice
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Spinning disk confocal microscopy was performed using an Olympus BX51 (Olympus, Center Valley, PA) upright microscope. The microscope used a confocal light path (WaveFx, Quorum) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation). Laser excitation at 488, 561 and 647 was used in rapid succession and fluorescence in green, red and far red channels was visualized with long pass filters (Semrock). Exposure time and sensitivity setting were uniformly maintained for each set of experiments. For imaging, velocity acquisition software (Improvision Inc. Lexington, KY) was used to drive the microscope. A 512×512 pixel back thinned EMCCD camera (C9100-13 Hamamatsu) was used for fluorescence detection. Mice were imaged using a 4×/0.16 air objective (Olympus, Center Valley, PA). Some mice were also imaged as required following 2 hours of infection with a 10×/0.30 numerical aperture air objective (Olympus, Center Valley, PA).
5
Intravital Imaging of Immune Cells
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Image acquisition was started 1 hour after APAP injection using an inverted spinning-disk confocal microscope (IX81; Olympus), equipped with a focus drive (Olympus) and a motorized stage (Applied Scientific Instrumentation). For laser microscopy, the microscope was linked with a confocal light path (WaveFx; Quorum Technologies) based on a modified CSU-10 head (Yokogawa Electric Corporation). Cells of interest (neutrophils, T cells, or KCs) were visualized using fluorescently conjugated antibodies, and CXCR6 was visualized by using gfp knockin reporter animals. Image recording and analysis were performed using Volocity software (PerkinElmer) and Imaris 7.7 (Bitplane).
6
Imaging Leukocyte Transmigration by Confocal Microscopy
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Spinning disk confocal microscopy was performed using an Olympus BX51W1 base (Olympus, Center Valley, PA) fitted either with 10x/0.30 UPlanFLN air or with10x/0.5 SFluor air, and 20x/0.95 XLUMPlanFI water immersion objectives. The microscope was equipped with a confocal light path (WaveFx, Quorum, Guelph, ON, Canada) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation, Tokyo, Japan). Mice were injected with antibodies immediately before imaging. Endothelial cells were stained with Alexa Fluor 647-conjugated anti-mouse PECAM-1 antibody (8 μg per mouse).
Neutrophils were stained with phycoerythrin (PE)-conjugated anti-mouse Ly-6G antibody (8 μg per mouse). Vascular transmigration was evaluated by exciting the fluorophores with two laser wavelengths (488 nm and 561 nm; Cobolt, Stockholm, Sweden) and visualizing with proper band-pass filters (Semrock, Rochester, NY). Volocity software (version 6.5.1, Improvision, Lexington, MA) was used to control the microscope, image acquisition, and analysis.
Neutrophils were stained with phycoerythrin (PE)-conjugated anti-mouse Ly-6G antibody (8 μg per mouse). Vascular transmigration was evaluated by exciting the fluorophores with two laser wavelengths (488 nm and 561 nm; Cobolt, Stockholm, Sweden) and visualizing with proper band-pass filters (Semrock, Rochester, NY). Volocity software (version 6.5.1, Improvision, Lexington, MA) was used to control the microscope, image acquisition, and analysis.
7
In Vivo Liver Metastasis Imaging
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Spinning disk confocal microscopy was performed on all animals from the liver metastasis model and splenectomized control mice using the customized Olympus IX81-inverted microscope for visualizing CT26-iRFP tumors and CXCR6-gfp þ cells in the liver. The microscope was equipped with an Olympus focus drive that had a motorized xyz stage (Applied Scientific Instrumentation, MS-2000 with piezo-z insert) and a motorized objective turret holding three objective lenses: 4Â/0.16 UPLANSAPO, 10Â/0.40 UPLANSAPO, and 20Â/0.70 UPLANSAPO. The objective lenses were connected to a light path (WaveFx; Quorum Technologies) that was linked to a Yokogawa CSU-10 head (Yokogawa Electric Corporation). The acquired images were recorded with an EM-CCD camera. The image acquisition software used was Volocity (Quorum Technologies). A detailed description of the procedure and preparation of the animals has been published in ref. 38 . For in vivo imaging of PD-1, PD-L1, and Kupffer cells in Cxcr6 gfp/þ knock-in mice, the following antibodies were used: anti-PD-1-APC (clone 29F.1A12, Biolegend), anti-PD-1-isotype control-APC (clone RTK2758, Biolegend), anti-PD-L1-PE (clone 10F.9G2, Biolegend), anti-PD-L1-isotype control-APC (clone RTK4530, Biolegend), and anti-F4/ 80-AF750 (clone BM8, AbLab).
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