Ixon3 897

Plasma membrane localization of WT and 3KQ mutant forms of C1-Ten were imaged using an objective-type TIRF microscope built on an inverted microscope (IX-81, Olympus, Tokyo, Japan) equipped with an XY-axis automated stage (MS-2000, Applied Scientific Instrumentation, Eugene, OR, USA). A 488-nm laser (35-LAL-415–220R, Melles Griot, NY, USA) was aligned and collimated to focus on the back focal plane of an oil-immersion 60× objective lens (APON 60×OTIRF/1.49, Olympus). A dichroic mirror (ZTUV-405/488/561RPC, Chroma, Taoyuan City, Taiwan) and an emission filter (ZET405/488/561M, Chroma) were used. Images were collected with an electron multiplying charge-coupled device (EM-CCD) camera (iXon3 897, Andor Technology, Belfast, UK). For higher magnification, we used a 1.6× amplifier and inserted a 1.43× tube lens. The images were obtained before and after insulin treatment (2 min, 5 min, 7 min, and 9 min) at a frame rate of 20 Hz. At the indicated time point, the fluorescence intensity of each cell was subtracted from the background fluorescence measured outside the cell. Fluorescence intensity was determined for each condition by normalizing fluorescence intensity of the image in the NT condition.

TIRF imaging was done using a Leica GSD/TIRFM ground state depletion superresolution microscope. Solid-state laser light (405, 488, 532, and 642 nm) was focused at the back focal plane of a 160× numerical aperture 1.43 objective. Signals were recorded by using an Andor iXon3 897 high-speed electron-multiplying charge-coupled device camera. For TIRF imaging, the stained cells were mounted in an open chamber filled with 1× PBS.

Imaging was performed on a Nikon Eclipse Ti inverted epifluorescence microscope using a 100× objective (Plan Fluo, NA 1.40, oil immersion) with a 2.5× TV relay lens, using a mercury lamp as the light source (X-Cite 200DC, Excelitas Technologies), within a cage incubator (InVivo Scientific) at 30 °C, and acquired using a cooled EMCCD (electron multiplying charge-coupled device) camera (iXon3 897, Andor, Belfast, United Kingdom). The fluorescent filters used in the study were as follows (Xnm, Yex [bandwidth] excitation filter/dichroic beamsplitter wavelength/Xnm, Yem [bandwidth] emission filter/company): cyan (436 nm, 20ex/455 nm/480 nm, 40em/Nikon), yellow (500 nm, 20ex/515 nm/535 nm, 30em/Nikon), and red (560 nm, 40ex/585 nm/630 nm, 75em/Nikon). The images are acquired with these exposures: phase-contrast (100 ms), red (500 ms), yellow (100 ms), and cyan (200 ms). These color names correspond to the filters used for the colors shown in the images.