2.5. Axonal transport imaging
About two days after transfection, the coverslips were cultured in a
Perfusion Chamber RC-26G (Warner Instruments, Hamden, CT) under an
upright fluorescence microscope (Suzhou NIR-Optics Technology Co., Ltd.,
China), the perfusion solution was a neuron culture medium, containing
Neurobasal media mixed with 1% penicillin-streptomycin, 1%
L-Glutamine, 2% B27 and 1% sodium pyruvate. The microscopic imaging
objective is a 60x/1.0 water lens. When operating, first use a low power
lens to locate the field of vision, and then a high power lens focuses
on the neuronal axon. A 575 nm excitation light from a SpectraX Lumencor
solid-state light source (Beaverton, OR, USA) was used to irradiate the
sample and the emitted light through a filter into the QImaging Retiga
R3 camera (Burnaby, BC Canada). Fluorescent images were acquired in TIFF
format by the Ocular 2.0 software that matched the camera and recorded
at the video mode with an exposure time of 500 ms. The recording
duration depended on the time of availing axonal transport observation.