Insights into metabolic heterogeneity of colorectal cancer gained from fluorescence lifetime imaging.

Heterogeneity of tumor metabolism is an important, but still poorly understood aspect of tumor biology. Present work is focused on the visualization and quantification of cellular metabolic heterogeneity of colorectal cancer using fluorescence lifetime imaging (FLIM) of redox cofactor NAD(P)H. FLIM-microscopy of NAD(P)H was performed in vitro in four cancer cell lines (HT29, HCT116, CaCo2 and CT26), in vivo in the four types of colorectal tumors in mice and ex vivo in patients' tumor samples. The dispersion and bimodality of the decay parameters were evaluated to quantify the intercellular metabolic heterogeneity. Our results demonstrate that patients' colorectal tumors have significantly higher heterogeneity of energy metabolism compared with cultured cells and tumor xenografts, which was displayed as a wider and frequently bimodal distribution of a contribution of a free (glycolytic) fraction of NAD(P)H within a sample. Among patients' tumors, the dispersion was larger in the high-grade and early stage ones, without, however, any association with bimodality. These results indicate that cell-level metabolic heterogeneity assessed from NAD(P)H FLIM has a potential to become a clinical prognostic factor.

Translucency and Color Stability of a Simplified Shade Nanohybrid Composite after Ultrasonic Scaling and Air-Powder Polishing.

We aimed to assess the influence of professional dental prophylaxis on the translucency and color stability of a novel simplified shade nanohybrid composite material. Sixty composite disks (5 mm in diameter and 2 mm thick) of light (n = 30) and dark (n = 30) shades were prepared. The specimens were randomly divided into the following three groups (n = 10) according to the prophylaxis procedure used: ultrasonic scaling, air-powder polishing with sodium bicarbonate, and controls. The specimens were submitted to translucency and color analysis based on the CIELab system. Two measurements were performed before and after 48-h storage in coffee. Translucency values of untreated light and dark specimens were 9.15 ± 0.38 and 5.28 ± 1.10, respectively. Air-powder polishing decreased the translucency of the light composite specimens. Storage in coffee resulted in color changes (∆E) ranging between 2.69 and 12.05 and a mean translucency decrease ranging between -0.88 and -6.91. The samples in the light group tended to exhibit greater staining; the treatment method had no effect on ∆E. It can be concluded that light-shade composite restorations are more prone to translucency and color changes resulting from air-powder polishing and contact with staining media. However, further research using other composites and powders is required.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria.

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10-5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.