Lambda 10 b smart shutter

For sensitized emission measurements of AKAR4 biosensor in HeLa cells wide-field images were captured with a Nikon TiE inverted microscope with a 20 × 0.5NA objective and a DS-Qi2 CMOS camera (Nikon, Japan). Images were acquired at intervals of 2 min with the Nikon NIS-Elements acquisition software using JOBS module (Nikon, Japan). A Lumencor Spectra X LED Light Engine (Lumencor, Beaverton, OR, USA) was used as excitation light source to reduce phototoxicity. Ratio imaging used a 440/30 nm excitation filter, a t440/510/575rpc multi-band dichroic mirror, and two emission filters (ET480/40 M (cyan fluorescent protein: CFP) and AT545/30 M (FRET)). Lumencor provided excitation filters, and all dichroic mirrors and emission filters were obtained from Chroma Technology (Brattleboro, VT, USA). An automated emission filter wheel Lambda 10-B Smart Shutter (Sutter Instrument, Novato, CA, USA) was used. The F480/F545 emission ratio, indicative of biosensor activation, calculated for each pixel on the whole image, was performed with custom routines written in IGOR Pro environment (Wavemetrics, Lake Oswego, OR, USA). Normalized F480/F545 emission ratio values were then plotted in PrismV (GraphPad software, La Jolla, CA, USA) and displayed in two ways: cell trace for each individual cell (grey) overlaid with the population mean in red and the 25^th and 75^th percentile values in blue.

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.