Plan fluor objective lens

Fixed cells were imaged using an inverted research widefield microscope (Eclipse Ti; Nikon) with perfect focus system, equipped with a 60×1.49 NA Apochromat total internal reflection fluorescence (oil) objective lens, a microscope cage incubator (OkoLab), and an EM charge-coupled device (CCD) camera (Andor Technology) controlled with NIS-Elements Ar 4.0 software (Nikon). All widefield images, unless specifically indicated otherwise were background subtracted by ImageJ's rolling ball (r=20) and sharpened for display by Fiji/ImageJ's unsharp mask filter (r=1, weight=0.6).
Fixed embryos were imaged on the same microscope using the Nikon C1 Confocal system. For time-lapse imaging, embryos were dechorionated at 50% epiboly stage and embedded in 0.3% agarose in E3 embryo medium. DIC images were taken every 15 min on the same microscope using a 10×0.3 NA Plan Fluor objective lens (Nikon). Embryo staging was determined by wild-type embryos imaged simultaneously with mutants or morphants. Live embryos at 12-24 hpf and fixed embryos after whole-mount in situ hybridization were imaged using a Leica MZFLIII upright microscope.

Embryos were dechorionated at 50% epiboly stage and embedded in 0.3% agarose in E3 embryo medium. DIC images of the lateral mesoderm during dorsal convergence (85-90% epiboly) were taken every 45 s on a Nikon Ti microscope using a 20×0.75 NA Plan Fluor objective lens. Cells residing in the layer closest to the yolk syncytium were tracked using Fiji/imageJ with the Manual Tracking plugin. Lateral mesodermal cells from wild-type embryos overexpressing α-catenin-GFP were imaged during dorsal convergence (85-90% epiboly) using a Leica SP8 confocal microscope. Images were taken every 45 s. Images were sharpened for display by using Fiji/ImageJ's Gaussian blur filter (r=2) and unsharp mask filter (r=2, weight=0.6).

Micro‐fluorescence images were obtained using a fluorescence microscope (TE2000‐U, Nikon, Melville, NY, United States) equipped with a 175 W full‐spectrum Xenon lamp, QuantEM EMCCD camera (512SC, Photometrics, Tucson, AZ, United States), NIR‐compatible optics (CY5.5 and CY7 Filter Set), an NIR‐compatible 10× Plan Fluor objective lens (Nikon), and DAPI filter set. Images were converted to pseudocolour (red/green) and analyzed by InnerView™ (INNERVIEW Co., Sungnam, Korea).
Macro‐fluorescence images were taken using a small animal in vivo imaging system (Pearl® Impulse, LI‐COR Biosciences, Lincoln, NE, United States), which was equipped with NIR channels (700 and 800 nm) and a white channel. The intensity of the NIR fluorescence signal from rats was monitored over the skin weekly for 8 weeks, while the rats were anesthetized with 3% isoflurane. In this study, the imaging system was remodeled to be used with rats (to avoid size limitations). Rats were sacrificed at 8 weeks for imaging after skin removal and histological analysis. Imaging data were collected and quantified by Pearl Software Images (LI‐COR Biosciences). The fluorescence region seen in the earliest sample was determined by the ROI. The mean of the fluorescence intensity (FL Mean; arbitrary unit) was presented in the results.