scholarly journals Label-free sensing of cells with fluorescence lifetime imaging: the quest for metabolic heterogeneity

2022 ◽  
Author(s):  
Evgeny A Shirshin ◽  
Marina V Shirmanova ◽  
Alexey V Gayer ◽  
Maria M Lukina ◽  
Elena E Nikonova ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Decay ◽  
Fluorescence Lifetime Imaging ◽  
Label Free ◽  
Biomedical Sciences ◽  
Lifetime Imaging ◽  
Endogenous Fluorophores ◽  
Novel Approach ◽  
Metabolic Heterogeneity ◽  
Decay Parameters

Molecular, morphological and physiological heterogeneity is the inherent property of cells, which governs differences in their response to external influence. The tumor cells metabolic heterogeneity is of a special interest due to its clinical relevance to the tumor progression and therapeutic outcomes. Rapid, sensitive and non-invasive assessment of metabolic heterogeneity of cells is of 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 (FDPs) of endogenous fluorophores, such as NAD(P)H. To achieve the accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this report, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity has been demonstrated. This has been achieved using a novel approach to data analysis based on the non-parametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations as compare more traditional approaches of FLIM measurements and analysis. The new approach was further validated for imaging cultured cancer cells treated with chemotherapy. Those results pave the way for an accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

scholarly journals Label-Free Macroscopic Fluorescence Lifetime Imaging of Brain Tumors

2021 ◽  
Vol 11 ◽  
Author(s):  
Maria Lukina ◽  
Konstantin Yashin ◽  
Elena E. Kiseleva ◽  
Anna Alekseeva ◽  
Varvara Dudenkova ◽  
...  
Keyword(s):  
White Matter ◽  
Brain Tumors ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Label Free ◽  
Time Resolved ◽  
Lifetime Imaging ◽  
Endogenous Fluorophores ◽  
Optical Markers ◽  
Brain Tissues

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients’ glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas.

scholarly journals Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging

2019 ◽  
Vol 20 (22) ◽  
pp. 5565 ◽  
Author(s):  
Leben ◽  
Köhler ◽  
Radbruch ◽  
Hauser ◽  
Niesner
Keyword(s):  
Fluorescence Lifetime ◽  
Metabolic Activity ◽  
Cellular Metabolism ◽  
Fluorescence Lifetime Imaging ◽  
Label Free ◽  
Analysis Framework ◽  
Cell Nuclei ◽  
Lifetime Imaging ◽  
Subcellular Resolution ◽  
In Cells

In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.

scholarly journals Three-Channel Fluorescence Lifetime Imaging of Endogenous Fluorophores

2016 ◽  
Vol 110 (3) ◽  
pp. 649a
Author(s):  
Zdenek Svindrych ◽  
Horst Wallrabe ◽  
Shagufta Rehman ◽  
Meghan J. O'Melia ◽  
Ammasi Periasamy
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Endogenous Fluorophores
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