Imaging Neuronal ATP Levels and Mitochondrial Transport

Transduced cortical neurons were transferred to chambers containing pre-warmed imaging buffer (Hibernate E low fluorescence medium (BrainBits) with 2% B27 and 0.5 mM GlutaMAX). A Zeiss LSM 880 Airyscan confocal microscope with a 40 × 1.3 NA oil immersion objective was used for live neuron imaging. To assess cellular ATP levels, images were collected at emissions 505–550 nm and above 545 nm within cell bodies or axons expressing GO-ATeam2 to measure ratiometric values which were generated in ImageJ (Huang et al. 2021 (link)). To analyze axonal mitochondrial transport, live images were captured along the microgrooves with 5-sec intervals, and kymographs were generated using ImageJ (Kang et al. 2008 (link)).