Optical microscopic imaging offers opportunities to perform noninvasive assessments of numerous parameters associated with the biochemistry, morphology, and functional state of biological samples. For example, it is possible to detect the endogenous fluorescence from a small number of important biomolecules, including NADH and FAD, which are two coenzymes involved in key metabolic pathways such as glycolysis, the Krebs cycle, and oxidative phosphorylation. Here, we review different imaging approaches to isolate the fluorescence from these chromophores in two- and three-dimensional samples and discuss the origins and potential interpretation of the observed signals in terms of cell metabolic status. Finally, we discuss the challenges and limitations of these approaches, as well as important research directions that we expect will evolve in the near future.

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.

Future work is needed to evaluate whether a combination of different biochemical and structural outcomes using these imaging techniques can provide complementary information regarding the utilization of specific metabolic pathways. Antioxid. Redox Signal. 00, 000-000.

A major limitation to cardiac tissue engineering and regenerative medicine strategies is the lack of proliferation of postnatal cardiomyocytes. The extracellular matrix (ECM) is altered during heart development and studies suggest it plays an important role in regulating myocyte proliferation. Here, we studied the effects of fetal, neonatal, and adult cardiac ECM on the expansion of neonatal rat ventricular cells in vitro. At 24 hr, overall cell attachment was lowest on fetal ECM; however ∼80% of the cells were cardiomyocytes while many non-myocytes attached to older ECM and poly-L-lysine controls. After 5 days, the cardiomyocyte population remained highest on fetal ECM, with a 4-fold increase in number. Significantly more cardiomyocytes stained positively for the mitotic marker phospho-histone H3 on fetal ECM compared to other substrates at 5 days, suggesting that proliferation may be a major mechanism of cardiomyocyte expansion on young ECM. Further study of the beneficial properties of early developmental aged cardiac ECM could advance the design of novel biomaterials aimed at promoting cardiac regeneration.

Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio are associated with enhanced levels of glycolysis relative to oxidative phosphorylation, increases in glutamine consumption to support energy production are associated with increased optical redox ratio values. Such mechanistic insights in the origins of optical metabolic assessments are critical for exploiting fully the potential of such noninvasive approaches to monitor and understand important metabolic changes that occur in live tissues at the onset of cancer or in response to treatment.