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Quantitative imaging of membrane micropolarity in living cells and tissues by spectral phasors analysis.

ABSTRACT: Intracellular micropolarity is essential in several metabolic processes, as it controls membrane permeability, regulating the fluxes of molecules and energy. Here we describe a method for the determination of the micropolarity in living cells using spectral confocal microscopy. The method is based on a phasor analysis of spectrally resolved images of live cells, labelled with the solvatochromic probe Nile Red. An application is provided to extract a polarity profile from the acquired Spectral datasets, which represent the contribution of hyperpolar, polar and non-polar lipids, and to generate a micropolarity map at submicrometric spatial resolution. A metabolic parameter, representing a quantitative index of the fatty acid-triacylglycerol turnover, is also furnished. This method allows a functional profiling of cells and tissues and the detection of metabolic imbalances between lipid storage and usage. •Use of spectral resolved confocal microscopy of Nile Red labelled cells for pixel resolved determination of the membranes micropolarity.•Spectral acquisition increases the specificity and sensitivity of the detection to provide a polarity profile and a metabolic index for fatty acid-TG turnover.•Use of spectral resolved confocal microscopy of Nile Red labelled cells for pixel resolved determination of the membranes micropolarity.

SUBMITTER: Di Giacinto F

PROVIDER: S-EPMC6232651 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Quantitative imaging of membrane micropolarity in living cells and tissues by spectral phasors analysis.

Di Giacinto Flavio F De Angelis Claudio C De Spirito Marco M Maulucci Giuseppe G

MethodsX 20181029

Intracellular micropolarity is essential in several metabolic processes, as it controls membrane permeability, regulating the fluxes of molecules and energy. Here we describe a method for the determination of the micropolarity in living cells using spectral confocal microscopy. The method is based on a phasor analysis of spectrally resolved images of live cells, labelled with the solvatochromic probe Nile Red. An application is provided to extract a polarity profile from the acquired Spectral da ...[more]

PMID: 30456174

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Project description:Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.

Dataset Information

Quantitative imaging of membrane micropolarity in living cells and tissues by spectral phasors analysis.

Publications

Quantitative imaging of membrane micropolarity in living cells and tissues by spectral phasors analysis.

Similar Datasets

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