Plan apo λ

Fluorescence and time-lapse microscopy was performed with an inverted fluorescence microscope (Nikon Ti-E, Nikon) equipped with an incubation chamber, an Orca Flash 4 digital camera [Hammamatsu a pE-100-LED (CoolLED)] as the transmission-light source, a Spectra X (Lumencor) as the fluorescent-light source and a 10× objective (Plan Apo λ; numerical aperture, 0.45; DIC N1; working distance, 4). Bright-field images (3% intensity, 90-ms exposure), d2EYFP fluorescence images (excitation, 513/17 nm; intensity, 50%; exposure, 200 ms; YFP ET filter, dichroic 520 nm; emission, 543/22 nm), and mCherry fluorescence images (excitation, 549/15 nm; intensity, 50%; exposure, 200 ms; CY3 HC, dichroic 562 nm; emission, 593/40) were collected. A binning of 2 × 2 was used.

Fluorescence microscopy was performed with an inverted fluorescence microscope (Nikon Ti-E; Nikon) equipped with an incubation chamber, an Orca Flash-4 digital camera (Hamamatsu), a pE-100-LED (CoolLED) as the transmission light source, a Spectra X (Lumencor) as the fluorescent light source, and a 10× objective (Plan Apo λ; numerical aperture, 0.45; DIC N1; working distance, 4). We collected bright-field images (40% intensity; 10 ms exposure), Dendra2 fluorescence images (before activation) (excitation, 490 nm; intensity, 10%; exposure, 200 ms; GFP ET filter, dichroic 495 nm; emission, 525/50 nm); Dendra2 (after activation) and mCherry fluorescence images (excitation, 553 nm; intensity, 40%; exposure, 500 ms; CY3 HC, dichroic 562 nm; emission, 593/40 nm); Citrine fluorescence images (excitation, 470 nm; intensity, 15%; exposure, 200 ms; YFP ET filter, dichroic 520 nm; emission, 543/22 nm); Fast-FT (blue variant) fluorescence images (excitation, 402 nm; intensity, 15%; exposure, 200 ms; DAPI HC, dichroic 416 nm; emission, 460/50 nm), and Fast-FT (red variant) fluorescence images (excitation, 553 nm; intensity, 40%; exposure, 500 ms, CY5 HC, dichroic 660 nm; emission, 700/75). A binning of 2 × 2 was used. Image analysis was performed with Imaris (Bitplane).

3D images were acquired with inverted wide-field microscope (Nikon Eclipse Ti) controlled by NES Elements v4.3 imaging software (Nikon) using a 60X/1.40 oil objective (Nikon Plan Apo λ), LED illumination (Lumencor) and CoolSNAP MYO CCD camera. Image acquisition, deconvolution and analysis was completed with NES Elements v4.3 imaging software. Only linear image corrections in brightness or contrast were completed. For all analyses, three-dimensional images of randomly selected fields were acquired and image acquisition parameters were kept constant for all the cover slips from the same experiment.