Intensilight c hgfi 130 w mercury lamp

Fluorescence microscopy images were acquired with a Nikon Eclipse 80i microscope and 10x/0.30 Nikon objective (Nikon Corporation), equipped with a SPOT RT3 CCD camera (Diagnostic Instruments), controlled by SPOT Advanced imaging software (v. 5.0) with Peripheral Devices and Quantitative Imaging modules. A Nikon Intensilight C‐ HGFI 130‐W mercury lamp, shuttered with a Lambda 10‐B SmartShutter (Sutter Instruments), was used for GFP excitation, and a GFP filter set (470/40 × 495lpxr 525/50 m; Chroma Technologies) was used for detection.

Live imaging was performed on a spinning disk confocal system that uses a Nikon 60 × 1.2 NA water objective, a Photometrics Prime 95B EMCCD camera, and a Yokogawa CSU-X1 confocal scanner unit. Images were acquired and analyzed by Nikon’s NIS imaging software and ImageJ/FIJI Bio-formats plugin (National Institutes of Health) (Linkert et al., 2010 (link); Schindelin et al., 2012 (link)). GCaMP3 images were also acquired by a 60×/1.40 NA oil-immersion objective on a Nikon Eclipse 80i microscope equipped with a SPOT RT39M5 sCMOS camera (Diagnostic Instruments, Sterling Heights, MI, USA) with a 0.63x wide field adapter, controlled by SPOT Advanced imaging software (v. 5.0) with Peripheral Devices and Quantitative Imaging modules. Images were acquired at 2448 × 2048 pixels, using the full camera chip, and saved as 8-bit TIFF files. Fluorescence excitation was provided by a Nikon Intensilight C-HGFI 130 W mercury lamp and shuttered with a Lambda 10-B SmartShutter (Sutter Instruments, Novato, CA), also controlled through the SPOT software. Single-channel GCaMP time-lapse movies were acquired using a GFP filter set (470/40 × 495 lpxr 525/50 m) (Chroma Technologies, Bellows Falls, VT) at 1 frame per second, with an exposure time of 40–60 ms, gain of 8, and neutral density of 16.