Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation

Abstract

The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

“…25 NADH quantification through the redox ratio has also been employed to determine stem cell differentiation. 26 In the cells' cytoplasm, a combination of freely diffusing and bound NADH is found. When excited at the correct wavelength, and in the absence of other exogenous or exotic fluorescent components, NADH is the main autofluorescent species that contributes to the optical emission, yielding a linear combination of the single exponential decays of free and bound NADH in the phasor plot.…”

Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

“…16,17 In order to test the robustness of the ORR for measuring cell metabolism, a number of studies have attempted to validate the correlation between ORR and the oxidation-reduction ratio of NAD þ ∕NADH. 18,19 Among them, Quinn et al 20 used liquid chromatography/mass spectrometry and found that while the fluorescence intensities of FAD þ and NADH are not correlated with their actual intracellular concentrations, the ORR is significantly correlated with NAD þ ∕NADH. However, no work has been reported to evaluate the stability and accuracy of ORR in living, dynamic biological samples.…”

Invited Review Article: Advanced light microscopy for biological space research

“…Autofluorescent cellular components (e.g., chlorophyll, NADH, collagen, and lipofuscins) do not require any extraneous markers and serve as reliable natural cell state indicators, since the emission often changes with cellular metabolism or in response to external and internal impulses. [42][43][44] The obvious advantage of autofluorescent molecules is their non-invasive character, but unfortunately, only few such molecules exist. Hence, cellular components are most often labeled.…”