Microscopy Techniques for Cellular Imaging

Brightfield images in Figure 2 and Figure 2—figure supplement 1 were imaged on a Hamumatsu Nanozoomer XR with ×20 and ×40 objectives. Macroscope images in Figure 1 and Figure 2 were imaged on a Nikon AZ100 Macroscope. Figure 1—figure supplement 3 was imaged on Leica Stellaris DMI8 equiped with 4 (HyD X/HyD S) GaSP detectors with ×40 or ×60 oil objectives. Fluorescent images in Figure 2, Figure 3A, Figure 3—figure supplement 1A, B, Figure 3—figure supplement 2A, Figure 3—figure supplement 3, and Figure 7—figure supplement 1 were taken on a Nikon A1+Confocal with Oil 60 or ×100 objectives with 405, Argon 561 and 640 lasers and GaSP detectors. Fluorescent images in Figure 1, Figure 2—figure supplement 1D, Figure 4D, E, K, Figure 4—figure supplement 1, and Figure 6—figure supplement 1 were taken with Andor Dragonfly and Mosaic Spinning Disc confocal. Images in Figure 3B, O, Figure 3—figure supplement 1C, H, I, Figure 3—figure supplement 2C–E, and Figure 6E, F were taken with Nikon SORA with 405 nm 120 mW, 488 nm 200 mW, and 561 nm 150 mW lasers, ×100 1.35 NA Si Apochromat objective and a Photometrics Prime 95B 11 mm pixel camera. High-speed video microscopy was performed on a Nikon Ti microscope with a ×60 Nikon Plan Apo VC ×60/1.20 water immersion objective, and Prime BSI, A19B204007 camera, imaged at 250 fps. 3D-SIM imaging in Figure 4B, C, H, Figure 5, Figure 5—figure supplement 1, Figure 6A, B, Figure 7, and Figure 8 was performed using the GE Healthcare DeltaVision OMX-SR microscope equipped with the ×60/1.42 NA oil-immersion objective and three cMOS cameras. Immersion oil with refractive index of 1.518 was used for most experiments, and z stacks of 5–6 µm were collected every 0.125 µm. Images were reconstructed using GE Healthcare SoftWorx 6.5.2 using default parameters. Images for quantifications were collected at the widefield setting using the same microscope. Figure 8—figure supplement 1 was imaged using a DeltaVision Elite high-resolution imaging system equipped with a sCMOS 2048x2048 pixel camera (GE Healthcare). Z-stacks (0.2 μm step) were collected using a ×60 1.42 NA plan apochromat oil-immersion objective (Olympus) and deconvolved using softWoRx (v6.0, GE Healthcare).

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Hall E.A., Kumar D., Prosser S.L., Yeyati P.L., Herranz-Pérez V., García-Verdugo J.M., Rose L., McKie L., Dodd D.O., Tennant P.A., Megaw R., Murphy L.C., Ferreira M.F., Grimes G., Williams L., Quidwai T., Pelletier L., Reiter J.F, & Mill P. (2023). Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis. eLife, 12, e79299.

Publication 2023

Argon Cmos Dragonfly Immersion Microscope Supplement Video microscopy

Corresponding Organization :

Other organizations : MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, University of California, San Francisco, Sinai Health System, Lunenfeld-Tanenbaum Research Institute, Universitat de València, Universitat Jaume I, Edinburgh Cancer Research, University of Toronto, Chan Zuckerberg Initiative (United States)

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Variable analysis

independent variables

Microscope type (Hamamatsu Nanozoomer XR, Nikon AZ100 Macroscope, Leica Stellaris DMI8, Nikon A1+Confocal, Andor Dragonfly and Mosaic Spinning Disc confocal, Nikon SORA, Nikon Ti)
Objective type and magnification (×20, ×40, ×60 oil, ×100)
Laser wavelengths (405 nm, Argon 561 nm, 640 nm)

dependent variables

Brightfield and fluorescent images

control variables

Refractive index of immersion oil (1.518)
Z-stack step size (0.125 μm, 0.2 μm)

controls

No positive or negative controls were explicitly mentioned.

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