Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells

Rück, Angelika; Hauser, Carmen; Mosch, S.; Kalinina, Sviatlana

doi:10.1117/1.jbo.19.9.096005

Cited by 53 publications

(21 citation statements)

References 46 publications

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“…While analysis of several FLIM parameters provides insights into physiological events in cells and tissues for these intrinsically fluorescent coenzymes, the key indicators are described in Table 1. The cascade of reactions involving several enzymes, changing NAD+/NADH, NADP +/NADH, and FADH2/FAD ratios and the NAD(P)H/FAD intensity REDOX ratio, [42][43][44] as well as the recently suggested preferred REDOX ratio measurement, fluorescence lifetime redox ratio (FLIRR), 34 can be tracked by these FLIM parameters. The intensity fractions a1 and a2 are the pre-exponential parameters associated with the shorter (τ1) and longer (τ2) lifetime components of a biexponential fluorescence decay model.…”

Section: Introductionmentioning

confidence: 99%

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

2020

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Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)-essential coenzymes in cellular respiration-have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

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Section: Introductionmentioning

confidence: 99%

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

2020

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“…Importantly, fluorescence lifetime is independent of fluorophore concentration, which makes it a valuable tool for quantitative studies in scattering and absorbing samples. Both frequency domain and time domain FLIM methods have been applied 14 – 16 . This manuscript uses the latter, also called Time-Correlated Single Photon Counting (TCSPC) 17 .…”

Section: Introductionmentioning

confidence: 99%

Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM

Wallrabe

Švindrych

Alam

et al. 2018

Sci Rep

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Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses.

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“…Based on our FLIM method for quantitative analysis of mitochondrial energy metabolism, we differentiate between the (1) Pre-Apoptotic, (2) Responsive and (3) Slow responder PCa cells. Previous studies have utilized NAD(P)H and FAD lifetime changes for the evaluation of metabolic variations in cancer 34 – 36 . For the first time, we demonstrate that along with NAD(P)H and FAD lifetime changes, Trp-quenching due to Trp-NAD(P)H FRET interactions, and E% vs NAD(P)H-a 2 %/FAD-a 1 % median correlation are sensitive parameters in predicting the drug response of PCa cells.…”

Section: Introductionmentioning

confidence: 99%

Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay

Alam

Wallrabe

Švindrych

et al. 2017

Sci Rep

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Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment. We have used Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify mitochondrial metabolic response in PCa cells by tracking auto-fluorescent NAD(P)H, FAD and tryptophan (Trp) lifetimes and their enzyme-bound fractions as markers, before and after treatment with anti-cancer drug doxorubicin. A 3-channel FLIM assay and quantitative analysis of these markers for cellular metabolism show in response to doxorubicin, NAD(P)H mean fluorescence lifetime (τm) and enzyme-bound (a2%) fraction increased, FAD enzyme-bound (a1%) fraction was decreased, NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio and ROS increased, followed by induction of apoptosis. For the first time, a FRET assay in PCa cells shows Trp-quenching due to Trp-NAD(P)H interactions, correlating energy transfer efficiencies (E%) vs NAD(P)H-a2%/FAD-a1% as sensitive parameters in predicting drug response. Applying this FLIM assay as early predictor of drug response would meet one of the important goals in cancer treatment.

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Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells

Cited by 53 publications

References 46 publications

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength

Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM

Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay

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