Opterra swept field confocal microscope

Human-induced pluripotent stem-cell-derived ventricular cardiomyocytes (iPSC-CMs, Axol Biosciences, Cambridgeshire, UK) were plated on fibronectin-coated glass-bottomed chamber slides and maintained as monolayers for eight days at 37°C, 5% CO₂ in a humidified atmosphere in Cardiomyocyte Maintenance Medium (Axol Biosciences, Cambridgeshire, UK). Cells were loaded with the fluorescent calcium indicator Fluo-4AM in Hanks balanced salts solution using the Fluo-4 calcium imaging kit according to manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA). After 15 min equilibration in the microscope stage incubator (Okolab, Burlingame, CA) 20X image sequences were acquired at a rate of 400 fps on an Opterra swept-field confocal microscope (Bruker, Middleton, WI) equipped with a 510–520 nm emission filter, an Evolve Delta 512 × 512 EMCCD digital monochrome detector (Photometrics, Tucson, AZ), and a Helios 488 nm solid state laser source (Coherent, Santa Clara, CA).

For live confocal imaging, embryos were anesthetized in 0.02 % tricaine and mounted in 1 % low melting agarose in 10 mM HEPES E3 medium as described [38 ]. Live high speed imaging of endosomal trafficking was performed on a Bruker Opterra swept field confocal microscope (Bruker Nano Surfaces FM, Middleton, WI) equipped with a Nikon CFI Plan Apo VC 60x oil immersion objective (NA 1.40). Embryos were 23 hpf at the beginning of the experiment, and 1–20 1-μm optical sections were captured every 2 s for a total duration of 400 s.

FRAP assays were carried out on a Bruker Opterra swept field confocal microscope, equipped with an enclosure box at 5% CO₂ and imaging and objective heater at 37°C. NIH3T3 cells were imaged in phenol red-free standard growth media. For assays involving ionomycin, standard growth media was replaced immediately prior to imaging with phenol red- and serum-free media supplemented with 0.083% DMSO (control), or DMSO with 2.5 µM ionomycin (#9995, CST). Image acquisition was performed with 60x/1.4NA/Oil objective lens (CFI Plan Apo Lambda) with a 30 µm pinhole array and 70 µm width slit. Fluorescence was recorded with a 5 s baseline followed by a complete photobleaching of a cytoneme with 488 nm and 561 nm lasers. Fluorescence recovery was recorded for 120 s with 100 ms exposure per channel with frames taken every 277 ms. Fluorescence recovery of individual regions of interest (ROI) along the cytoneme to its tip were normalized with pre-FRAP equal to 100% and post-FRAP equal to 0%. FRAP curves were corrected for any loss of fluorescence during acquisition (Fritzsche and Charras, 2015 (link)).