Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue

Abstract

We describe a label-free imaging method to monitor stem-cell metabolism that discriminates different states of stem cells as they differentiate in living tissues. In this method we use intrinsic fluorescence biomarkers and the phasor approach to fluorescence lifetime imaging microscopy in conjunction with image segmentation, which we use to introduce the concept of the cell phasor. In live tissues we are able to identify intrinsic fluorophores, such as collagen, retinol, retinoic acid, porphyrin, flavins, and free and bound NADH. We have exploited the cell phasor approach to detect a trend in metabolite concentrations along the main axis of the Caenorhabditis elegans germ line. This trend is consistent with known changes in metabolic states during differentiation. The cell phasor approach to lifetime imaging provides a label-free, fit-free, and sensitive method to identify different metabolic states of cells during differentiation, to sense small changes in the redox state of cells, and may identify symmetric and asymmetric divisions and predict cell fate. Our method is a promising noninvasive optical tool for monitoring metabolic pathways during differentiation or disease progression, and for cell sorting in unlabeled tissues.phasor analysis | single cell metabolism T he hallmark of stem cells is their ability to produce a new stem cell by self-renewing, as they generate daughter cells that are committed to differentiation and form specialized tissues (1). The modulation of the balance between self-renewing divisions and differentiation is a central mechanism for stem cells during embryo development, adult tissue regeneration, and homeostasis. Stem-cell differentiation is a complex process mediated both by intrinsic molecular mechanisms and extrinsic signaling. Intrinsic cell polarity, subcellular localization mechanism, asymmetric centrosome and spindles, as well as cell-cycle regulators can establish self-renewing asymmetry during stem-cell division (2, 3). The influence of external chemical and physical stimuli, such as molecular gradients, extracellular matrix remodeling, and niche signaling is crucial for stem-cell plasticity and tissue development (4-6). Extrinsic signals can be propagated through intracellular signal-transduction pathways that converge to genetic networks that control pluripotency. Many different signaling and transcriptional pathways, which are important for development and cell-cycle regulation, converge on regulation of the redox state of stem cells (5,7,8). In turn, histone posttranscriptional modifications and epigenetic mechanisms, such as phosphorylation, acetylation, and methylation, sense cellular metabolism and the variation of metabolites levels (9). Hence, redox balance plays an important role in the maintenance and modulation of stem cell self-renewal and differentiation (10-12).In stem-cell research there is a high demand for noninvasive techniques to investigate self-renewal and differentiation. Methods, such as immunohistochemistry, metabolic assays, and PCR are currently...

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

“…Phasor fluorescence lifetime microscopy (FLIM) analysis proceeds by Fourier transformation of the lifetime data, which allows direct quantification at each pixel of the free and bound NADH ratio (34)(35)(36). Metabolic transitions and phenotypes in living single cells have been previously determined using the phasor approach to FLIM, including identification of biological processes, such as cell proliferation and differentiation, tumorigenesis, and aging (22,24,27,37,38). Because biochemical reactions depending on NADH occur at different rates in distinct subcellular compartments, the measurement of NADH free/bound ratio provides a weighted mean of the enzymatic activities in the cell when combined with local NADH fluorescence intensity.…”

“…Lifetime measurements from fluorescent NADH distinguish free NADH and subpopulations of protein-bound NADH, whereas NAD + is not fluorescent (21). NADH 2P-FLIM cellular map provides sensitive measurements of local activity associated with NADH metabolism (22)(23)(24)(25)(26)(27)(28). Fluorescence correlation spectroscopy analyzes the fluctuation of fluorescent molecules in a small illuminated spot, providing spatiotemporal maps of concentration, interaction, or diffusion parameters of molecules (29)(30)(31)(32)(33).…”

Cell Metabolomics

“…By using a combination of feature selection, pattern recognition, and multivariate data analysis approaches, metabolomic profiling aims to provide a comprehensive assessment of the alterations in the metabolite levels in cells (Lange et al, 2011;Stringari et al, 2011;Turnbaugh et al, 2011). Recent technological advancements in nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) have led to wide use of these technologies for precise measurements of metabolites with improved sensitivity, resolution, and mass accuracy (Fischer et al, 2012;Kleiner et al, 2012).…”

Reliable Cell Segmentation Based on Spectral Phasor Analysis of Hyperspectral Stimulated Raman Scattering Imaging Data

“…17 Later on, Gratton demonstrated its great potential in simplifying the analysis of FLIM and FRET images. 18,19 FLIM data collected in the time domain often exhibit complex nonexponential decay behavior. Simple exponential fitting is prone to error and requires expertise.…”