Characterizing Tissue Oxygen Tension During Neurogenesis in Human Cerebral Organoids
Year: 2025
Type: article
Abstract: Oxygen tension is a key regulator of early human neurogenesis; however, quantifying intra-tissue O2 in 3D models for an extended period remains difficult. Existing approaches, such as needle-type fiber microsensors and intensity-based oxygen probes or time-domain lifetime imaging, either perturb the organoids or require high excitation doses that limit the measurement period. Here, we present a step-by-step protocol to measure intra-organoid oxygen in human cerebral organoids (hCOs) using embedded ruthenium-based CPOx microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM). The workflow covers dorsal/ventral cerebral organoid patterning, organoid fusion at day 12 with co-embedded CPOx beads, standardized FD-FLIM acquisition (470-nm external modulation, 16 phases at 50 kHz, dual-tap camera), automated bead detection and lifetime extraction in MATLAB, and session-matched Stern–Volmer calibration with Ru(dpp)3(ClO4)2 to convert lifetimes to oxygen concentration. The protocol outputs per-bead oxygen maps and longitudinal patterns stratified by bead location (intra-organoid vs. gel) and sample state (healthy vs. abnormal), enabling direct linkage between developmental growth and oxygen dynamics.
Source: BIO-PROTOCOL
Authors: Yuan- Liu, Hsiao- Wu
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W4415497117