Orca flash 4.0 c11440 22c scmos camera

Chromaffin cells were imaged at room temperature using an inverted microscope (Ti-E, Nikon). TIRF microscopy was done using a MLC400 monolithic laser combiner (Agilent Technologies), ZT405/488/561/640rpc dichroic filter (Chroma), and Plan Apo Lambda 100× 1.45 N.A. oil objective (Nikon). Rapid FFN imaging was done using ET455/50 m emission filter (Chroma) and Orca Flash 4.0 C11440-22C sCMOS camera (Hamamatsu). Dual imaging of FFN and BDNF-pHluorin was done using ZET488/561/635m emission filter (Chroma) and iXon DU-897 EMCCD camera (ANDOR). Dual imaging of FFN with Alexa Fluor 488 (ThermoFisher) or CF488A (Biotium) was done using Dual-View filter cube (Optical Insights) mounted with T510lpxrxt beam-splitter (Chroma), ET460/36 m and ET545/40 m emission filters (Chroma), and Orca Flash 4.0 C11440-22C sCMOS camera (Hamamatsu). Confocal imaging of FFN and Alexa Fluor 488 was done using an integrated laser engine (ANDOR), Borealis CSU-W1 spinning disk (ANDOR), ZT405/488/561/640rpcv2 dichroic filter (Chroma), Plan Apo VC 100× 1.4 N.A. oil objective (Nikon), ZET405/488/561/635 emission filter (Chroma), and iXon DU-888 EMCCD camera (ANDOR). Cells were imaged in modified Tyrode's solution, and exocytosis was stimulated with 45 mM K⁺ by adding an equal volume of stimulation buffer (in mM, 54.5 NaCl, 10 HEPES-NaOH, pH 7.4, 10 glucose, 90 KCl, 5 CaCl₂, 1 MgCl₂).