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3D O2 imaging in the neuronal spheroids

Availability of O2 determines the cell metabolic status and physiological responses to stimulation. The multicellular spheroids made of cultured or primary neuronal cells are commonly used to study neural stem cell proliferation, differentiation and pathological states under conditions that resemble their native micro‐environment. The live cell imaging with cell‐penetrating phosphorescent O2‐sensitive probes allows quantification and high‐resolution mapping of O2 distribution in such structures. Using the Pt‐porphyrin nanoparticle (MM2, NanO2) and peptide‐based icO2 probes, we optimised conditions for internal (core) and surface staining of the rat primary neurospheres, to achieve high phosphorescent signals and low toxicity. Neurosphere oxygenation was analysed on FLIM and 2photon ratiometric‐based imaging systems. The observed gradients of neurosphere oxygenation were correlated with the O2 in the chamber and the localisation of stem cells in the core. Spectral characteristics of icO2 probes were compatible with live‐cell imaging fluorescent markers such as TMRM (mitochondrial membrane potential), BCECF (intracellular pH) or following fixation and immunofluorescence staining. Altogether, 3D O2 imaging represents versatile tool for high‐resolution assessment of metabolic, proliferation and differentiation status of neural stem cell in the spheroid culture. Supported by SFI.