Ixon ultra du 897 emccd camera

For total internal reflection fluorescence (TIRF) imaging we used a commercial TIRF system based on a Nikon Ti-E eclipse microscope equipped with a 100× Apo TIRF DIC oil immersion objective NA of 1.49 (Nikon Instruments Inc.), an iXon Ultra DU-897 EMCCD camera (Andor Technology Ltd.), and four main laser lines, 405 (Cube, Coherent Inc), 488 (Melles-Griot), 561 (Sapphire, Coherent Inc), and 640 (Cube, Coherent Inc) with corresponding filter sets. Isolated cells were fixed using 4% PFA and labelled with phalloidin using a buffer containing 0.05% saponin for 1 h.
For quantification of the grade of actin polymerization, an ImageJ plugin ridge detection was used to trace actin filaments, detected with phalloidin stain, in TIRF microscopy images. Standard values were used, and the threshold adjusted until most of the visible actin was traced. Images were exported using the “make binary” command. A region of interest (ROI) of roughly ¼ of the cell was chosen and used to obtain consistent data on the grade of polymerization. This ROI was used on all cells to obtain the area of binary cell traces within the threshold.

For TIRF imaging, we used a commercial TIRF system based on a Nikon Ti-E eclipse microscope equipped with a 100 × Apo TIRF DIC oil immersion objective NA of 1.49 (Nikon Instruments), an iXon Ultra DU-897 EMCCD camera (Andor Technology), and four main lasers, 405 nm (Cube, Coherent), 488 nm (Melles-Griot), 561 nm (Sapphire, Coherent), and 640 nm (Cube, Coherent) with corresponding filter sets. Isolated cells were fixed using 4% paraformaldehyde and labeled with antibodies in a buffer containing 1% BSA, 1% goat serum, and 0.05% saponin, 1 h per labeled antibody, and imaged as previously described (Wasserstrom et al., 2018 (link)). Puncta detection was performed to identify GSVs using Blob Finder (ZEISS Arivis software) at size cutoff set at <140 nm stepwise for each channel. The number of GSVs was determined by detection of puncta co-labelled with GLUT4 and IRAP. Signals detected within 40 nm distance were considered to arise from the same puncta.

Imaging was performed using a Nikon A1 plus confocal microscope with a 60x Apo DIC oil immersion objective with a NA of 1.40 (Nikon Instruments Inc.) and appropriate filter sets. Images were acquired with NIS-elements, version: 4.50.02, (Laboratory Imaging). For TIRF imaging we used a commercial TIRF system based on a Nikon Ti-E eclipse microscope equipped with a 100× Apo TIRF DIC oil immersion objective NA of 1.49 (Nikon Instruments Inc.), an iXon Ultra DU-897 EMCCD camera (Andor Technology Ltd.), and four main laser lines, 405 (Cube, Coherent Inc), 488 (Melles-Griot), 561 (Sapphire, Coherent Inc), and 640 (Cube, Coherent Inc) with corresponding filter sets. Isolated cells were fixed using 4% PFA and labelled with antibodies in a buffer containing 1% BSA, 1% goat serum and 0.05% saponin, 1–2 hours per labelled antibody. For neutral lipid staining BODIPY was used in conjunction with confocal imaging. TIRF microscopy was used to detect protein stain only. For imaging of adipocytes, we used previously described protocol^{52 (link)}.