Csu x1 spinning disk confocal

PtK2 GFP-α-tubulin cells were plated on 35 mm #1.5 coverslip glass-bottom dishes coated with poly-D-lysine (MatTek, Ashland, MA) and imaged in CO₂-independent MEM (Thermo Fisher). Cells were maintained at 27–32°C in a stage top incubator (Tokai Hit, Fujinomiyashi, Japan), without a lid. Live imaging was performed on a CSU-X1 spinning-disk confocal (Yokogawa, Tokyo, Japan) Eclipse Ti-E inverted microscopes (Nikon) with a perfect focus system (Nikon, Tokyo, Japan), and included the following components: head dichroic Semrock Di01-T405/488/561, 488 nm (150 mW) and 561 (100 mW) diode lasers (for tubulin and microneedle respectively), emission filters ETGFP/mCherry dual bandpass 59022 M (Chroma Technology, Bellows Falls, VT), and Zyla 4.2 sCMOS camera (Andor Technology, Belfast, United Kingdom). Cells were imaged via Metamorph (7.10.3, MDS Analytical Technologies) by fluorescence (50–70ms exposures) with a 100×1.45 Ph3 oil objective through a 1.5 X lens, which yields 65.7 nm/pixel at bin = 1.

Unless otherwise indicated, all fluorescence images shown in this article represent maximum projection images derived from a z-stack.
To acquire images of fixed dissociated cultures or brain sections we used an Olympus IX-70 microscope equipped with a CSU-X1 spinning disk confocal (Yokogawa Electric Corporation) custom equipped with 405 nm, 491 nm, 561 nm and 640 nm 50 mW solid state lasers (Solamere Technology Group Inc.) and a CoolSNAP HQ2 digital CCD camera (Photometrics) with pixel size of 91 nm. Fluorescence emission was selected through the following bandpass filters: 525/50 nm, 595/50, 700/75. Metamorph (Molecular Devices) was used to acquire a stack of 6–11 images in the z-dimension using optical slice thickness of 0.2 µm for 60X images of dissociated cultures and brain sections. A single plane of focus was used to acquire low magnification images of brain sections using a ×1.25 objective.