Gm 8000

Live cell imaging was performed on an epifluorescence microscope (Leica DMI6000B) equipped with an on-stage CO₂ incubation chamber (Tokai Hit GM-8000) and a motorized stage (Prior). An adaptive focus control was used to actively keep the image in focus during the period of imaging. A light-emitting diode (LED) light source (Lumencor Sola) was used as the fluorescence light source. For blue-light stimulation, pulsed blue light (200 ms pulse duration at 9.7 W/cm²) was used for GFP imaging and to initiate CRY2-CIB1 and CRY2-CRY2 interactions. For mCherry imaging, pulsed green light (200 ms pulse duration) was used. Fluorescence signal from GFP was detected using a commercial GFP filter cube (Leica; excitation filter 472/30, dichroic mirror 495, emission filter 520/35); fluorescence signal from mCherry was detected using a commercial Texas red filter cube (Leica; excitation filter 560/40, dichroic mirror 595, emission filter 645/75). The images for ERK or AKT translocation were acquired with an oil-immersion 100× objective, while the images for PC12 or DRG morphologies were acquired with 10× or 40× objectives. All experiments were imaged with a sensitive CMOS camera (PCO.EDGE 5.5) (PCO).

Prior to imaging, the culture medium was changed to an imaging medium derived from ref. ^{23 (link)} containing increased glucose to prevent starvation-induced filopodia:^{45 (link)} 20 mM HEPES at pH 7.5, 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 4.5 g/L glucose supplemented with 10% fetal bovine serum. Live cell imaging was performed on an epifluorescence microscope (Leica DMI6000B) equipped with an on-stage CO2 incubation chamber (Tokai Hit GM-8000) and a motorized stage (Prior). An adaptive focus control was used to actively keep the image in focus during the period of imaging. A light-emitting diode (LED) light engine (Lumencor) was used as the light source for fluorescence imaging. Pulsed blue light (200 ms pulse duration at 5.3 W/cm²) was delivered every 10 s both to image GFP-tagged constructs and to initiate iLID/SspB interactions. Pulsed green light (200 ms pulse duration) was used to image mCherry or tdTomato. The microscope was equipped with a commercial GFP filter cube (Leica; excitation filter 472/30, dichroic mirror 495, emission filter 520/35) and a commercial Texas red filter cube (Leica; excitation filter 560/40, dichroic mirror 595, emission filter 645/75). All Images were acquired with an oil-immersion 100× objective. All experiments were imaged with a sensitive CMOS camera (PCO.EDGE 5.5) (PCO).