Visualization of epifluorescence signals was performed as described previously (Hartmann et al., 2001 (link)). Briefly, cover slips with transfected cells were transferred into Petriperm dishes (Vivascience) with folio bottom and inspected with an inverted microscope (Olympus IX70) using 40 × (N.A.: 1.0) and 100 × (N.A.: 1.35) oil immersion objectives. Image capture was performed using a cooled CCD camera (Sensys 1401E, Photometrics), controlled by MetaVue software (Molecular Devices). Exposure times were chosen such that saturation was avoided. Processing of images was performed by MetaVue and Adobe Photoshop software without compromising the evident primary image information. However, in the figures, fluorescence in the soma and the proximal dendrites is often enhanced close to saturation in order to make single vesicles in distal neurites also clearly visible.
Some of the experiments were performed using an upright microscope (Zeiss, Axioscope FS) equipped with a 40 × objective (Olympus, LumPlanFI, N.A. 0.8 w). DAF-FM fluorescence was exited at 485 nm with a VisiChrome high speed monochromator (Visitron, Puchheim, Germany). Fluorescence images were taken with a Photometrics CoolSnap HQ2 CCD camera (Roper Scientific) at 0.1 Hz after passing a 510 nm long pass filter. Image acquisition and analysis was performed using Metafluor software (Molecular Devices).
Some of the experiments were performed using an upright microscope (Zeiss, Axioscope FS) equipped with a 40 × objective (Olympus, LumPlanFI, N.A. 0.8 w). DAF-FM fluorescence was exited at 485 nm with a VisiChrome high speed monochromator (Visitron, Puchheim, Germany). Fluorescence images were taken with a Photometrics CoolSnap HQ2 CCD camera (Roper Scientific) at 0.1 Hz after passing a 510 nm long pass filter. Image acquisition and analysis was performed using Metafluor software (Molecular Devices).