Cfi apochromat tirf objective

Confocal imaging was performed using a Perkin Elmer VoX-1000 Spinning Disk microscope equipped with a 60 ×/1.4 NA/oil CFI Apochromat TIRF objective, four laser lines (405, 488, 561 and 635 nm) and a Hamamatsu EMCCD camera (C9100-50). The following filter sets were used: excitation: quad-bandpass 405/488/568/640 nm with the matching emission filters for DAPI (445/30 nm), Alexa Fluor 488 (500–548 nm), TRITC (526–623 nm) and Cy5 (664–750 nm). For higher special resolution, images were acquired using a Leica SP5 II laser scanning microscope using a 100 × 1.44 NA HCX PL APO Objective with a pixel size of 86.6 nm and a z-spacing of 125 nm for subsequent deconvolution. For imaging the 405, 488, 561 and 633 nm laser line and spectral detection windows of 425–465 nm (DAPI), 495–558 nm (Alexa 488), 600–660 nm (Alexa 594) and 640–705 nm (Cy5) were used. Images were then deconvolved with wavelength specific point spread functions using ImageJ and the Iterative Deconvolution 3D plugin [58 ]. In addition, a Zeiss Axiovert 200 with a 100 ×/1.4 NA/oil Plan-Apochromat objective was used to image metaphase spreads. Images were recorded using a Zeiss Axiocam mRM, and the following filters were used: DAPI; ex: 350/50 nm; bs: 400 nm; em: 460/50 nm and Alexa Fluor 488: ex: 482/18 nm; bs: 495 nm; 520/28 nm.

Total internal reflection fluorescence (TIRF) microscopy was performed using a Nikon CFI Apochromat TIRF objective ( × 100, 1.49 NA) and sCMOS camera (ORCA-Flash4.0 V2, Hamamatsu, Japan) mounted on an Eclipse Ti-E inverted microscope with Perfect Focus System (PFS; Nikon). Imaging was performed on PTECs expressing STIM1, ORAI1-3, AMN, clathrin light chain and caveolin-1 tagged with cyan (CFP), enhanced yellow (EYFP) or mCherry fluorescent proteins as indicated. CFP, EYFP and mCherry were excitated by 405-, 488- and 561-nm lasers with corresponding filter lens, respectively. Colocalization analysis was performed with NIS-Elements AR v4.30 (Nikon) and 3D surface plot of fluorescent intensities was generated by Image-Pro Plus 6.0 (Media Cybernetics). Three to five experiments were performed for each condition.