Hc pl apo 20 0.75 cs2

Confocal imaging was performed using a commercial Leica SP8 TCS microscope (Leica Microsystems) equipped with four laser lines 405 nm, 488 nm, 552 nm and 638 nm. Samples within each independent experiment were acquired with equal settings. Images were acquired with an HC PL APO 20×/0.75 CS2 or HC PL APO 63×/1.40 CS2 (oil) objectives (Leica Microsystems), a scanning format of 1,024 × 1,024, eight-bit sampling, and 1 zoom, yielding a pixel dimension of 567.62 × 567.62 or 90.09 nm × 90.09 nm in the x and y dimensions, respectively.

Whole-mount tissue images were obtained using the TCS SP5 II or TCS SP8 confocal microscope equipped with HC PL APO 20×/0.75 CS2 and HC PL APO 63×/1.40 OIL CS2 objective lenses (Leica Microsystems). Specimens were scanned sequentially with a 405-nm diode laser, a 488-nm argon laser, a 543-nm He-Ne laser or a 561-nm diode laser, and a 633-nm He-Ne laser. Emission signals were collected by using a 440–480-nm emission filter for DAPI, a 504–538-nm emission filter for Alexa Flour 488, a 567–607-nm emission filter for Alexa Flour 555 or tdTomato, and a 658–780-nm emission filter for Alexa Flour 633 or 647. For image acquisition, 61.5–582 μm × 61.5–582 μm x–y planes were scanned at a resolution of 0.06–0.57 μm per pixel, and image stacks of 37–201 x–y planes with 0.5-μm z spacing were constructed after averaging 3–4 frames for each x–y plane.

For fluorescence lifetime imaging, 3.5 × 10⁴ MDCK cells were seeded in a Culture-Insert 2 well placed on a 24-well glass-bottom plate coated with 0.3 mg ml⁻¹ type I collagen. 24 h after seeding, the silicone confinement was removed, and the medium was exchanged for Medium 199 supplemented with 1% BSA, 100 unit mL⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin. 6 h after the removal of the silicone confinement, the cells were imaged to measure the fluorescence lifetime of Turquoise. Lifetime imaging was performed with HC PL APO 20×/0.75 CS2 under a Leica TCS-SP8 microscope (Leica Microsystems GmbH) equipped with a stage top incubator (Tokai Hit), a Lecia HyD SMD detector and a 440 nm ps pulsed diode laser (PDL 800-D; PicoQuant), which pulsed at a frequency of 80 MHz. The band path of emission wavelength was set from 450 to 485 nm. Time-lapse images were acquired every 10 min. The acquisition time for each measurement was 45 s. The amplitude-weighted mean fluorescence lifetimes were calculated in a pixel-by-pixel fashion using fitting with a multi-exponential (“n-exponential” function) reconvolution with adjustment of the number of components to two according to the manufacturer’s protocol (Leica Microsystems GmbH).