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3D O 2 imaging in the neuronal spheroids
Author(s) -
Dmitriev Ruslan I.,
Zhdanov Alexander V.,
Nolan Yvonne M.,
Papkovsky Dmitri B.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.574.1
Subject(s) - neurosphere , spheroid , neural stem cell , microbiology and biotechnology , stem cell , biophysics , cell , chemistry , live cell imaging , intracellular , fluorescence lifetime imaging microscopy , cell culture , biology , fluorescence , cellular differentiation , biochemistry , in vitro , adult stem cell , physics , genetics , quantum mechanics , gene
Availability of O 2 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 O 2 ‐sensitive probes allows quantification and high‐resolution mapping of O 2 distribution in such structures. Using the Pt‐porphyrin nanoparticle (MM2, NanO2) and peptide‐based icO 2 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 O 2 in the chamber and the localisation of stem cells in the core. Spectral characteristics of icO 2 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 O 2 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.