Journal: STAR Protocols
Article Title: Visualization and quantification of the stromal-vascular compartment in fetal or adult mouse bones: From sampling to high-resolution 3D image analysis
doi: 10.1016/j.xpro.2022.101222
Figure Lengend Snippet: Example settings for laser scanning confocal image acquisition
Article Snippet: The following equipment was used for the protocols explained in this manuscript: • Nikon TiE inverted C2 confocal microscope with the following specifications: ○ C2-DU3 detector ○ Plan Apo 10× objective ○ Plan Apo VC 20× objective ○ Diode lasers (measured downstream, in the focal plane of a Nikon Plan Apo VC 20× objective): - 405 nm (LU-N4 – 1.25 mW) - 488 nm (LU-N4 – 3.06 mW) - 561 nm (LU-N4 – 3.75 mW) - 640 nm (LU-N4 – 3.94 mW) ○ Emission filters: - Blue: 425–475 nm and 465–500 nm - Green: 500–550 nm, 515–550 nm, 525–555 nm, 500–530 nm - Red: 570–620 nm, 560–618 nm - Far-red: 663–738 nm • Nikon NiE upright spinning disk confocal microscope with the following specifications: ○ Camera (Teledyne Photometrics Prime 95B sCMOS): - 95% QE - 11 × 11 μm pixel size - 40 fps (16-bit) / 80 fps (12-bit) - 1.6e – read-noise ○ Stage (Luigs & Neumann 380FM-U shifting table + bridge 500): - 25 mm travel in XY - 2.5 nm motor resolution - Reproducibility less than 1 μm - Weight: 23 kg - Dimensions: 622 × 505 70 mm - Spindle lead 0.5 mm ○ Lasers (Agilent MLC400) (measured in the focal plane of a Nikon Plan Apo VC 20× objective): - 405 nm (diode – violet cube 0.23 mW) - 488 nm (blue diode – sapphire 1.83 mW) - 561 nm (green diode – sapphire 2.27 mW) - 640 nm (red diode – sapphire 1.76 mW) ○ Filters: - Quad cube: 432/515/595/730-25 nm - Blue 405 nm single: 450/50 nm - Green 488 single: 525/50 nm - Red 561 single: 595/50 nm - Far-red 640 nm single: 700/75 nm • Analysis PC (HP Z840) ○ Operating system: Windows 10 ○ RAM: 192 GB ○ Intel(R) CPU ES-2623 v3 @ 3.00 GHz 2.99 GHz (2 processors) #16 ○ NVIDIA Quadro RTX 4000 • Analysis PC (Dell precision tower 5810) ○ Operating system: Windows 10 ○ RAM: 64 GB ○ Intel(R) Xeon(R) CPU E5-1660 v3 @ 3.00 GHz - #16 ○ NVIDIA Quadro K4200 • Rocker / shaker – Biosan MR-12 • Heater / stirrer – IKA RCT basic
Techniques: Microscopy, Software, Refractive Index
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