Superlocalized Three-Dimensional Live Imaging of Mitochondrial Dynamics in Neurons Using Plasmonic Nanohole Arrays

We investigated the transport of neuronal mitochondria using superlocalized near-fields with plasmonic nanohole arrays (PNAs). Compared to traditional imaging techniques, PNAs create a massive array of superlocalized light beams and allow 3D mitochondrial dynamics to be sampled and extracted almost in real time. In this work, mitochondrial fluorescence excited by the PNAs was captured by an optical microscope using dual objective lenses, which produced superlocalized dynamics while minimizing light scattering by the plasmonic substrate. It was found that mitochondria move with an average velocity 0.33 ± 0.26 μm/s, a significant part of which, by almost 50%, was contributed by the movement along the depth axis ( z-axis). Mitochondrial positions were acquired with superlocalized precision (σ _x = 5.7 nm and σ _y = 11.8 nm) in the lateral plane and σ _z = 78.7 nm in the z-axis, which presents an enhancement by 12.7-fold in resolution compared to confocal fluorescence microscopy. The approach is expected to serve as a way to provide 3D information on molecular dynamics in real time.