Investigation of tryptophan–NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

Jyothikumar, Vinod; Sun, Yuansheng; Periasamy, Ammasi

doi:10.1117/1.jbo.18.6.060501

Cited by 29 publications

(19 citation statements)

References 9 publications

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“…Our work contributes to the growing evidence of glycolytic switch and increased optical redox ratio being common to a wide range of cancers. As tissue is a complex medium and fluorophores are known to interact with one another, diagnostic information from individual fluorophores can often be lost or misinterpreted [26]. Additionally, physiological changes such as tissue thickening or hardening may affect fluorescence measurements irrespective of fluorophore.…”

Section: Discussionmentioning

confidence: 99%

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

Palmer

Litvinova

Rafailov

et al. 2015

Biomed. Opt. Express

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The optical redox ratio as a measure of cellular metabolism is determined by an altered ratio between endogenous fluorophores NADH and flavin adenine dinucleotide (FAD). Although reported for other cancer sites, differences in optical redox ratio between cancerous and normal urothelial cells have not previously been reported. Here, we report a method for the detection of cellular metabolic states using flow cytometry based on autofluorescence, and a statistically significant increase in the redox ratio of bladder cancer cells compared to healthy controls. Urinary bladder cancer and normal healthy urothelial cell lines were cultured and redox overview was assessed using flow cytometry. Further localisation of fluorescence in the same cells was carried out using confocal microscopy. Multiple experiments show correlation between cell type and redox ratio, clearly differentiating between healthy cells and cancer cells. Based on our preliminary results, therefore, we believe that this data contributes to current understanding of bladder tissue fluorescence and can inform the design of endoscopic probes. This approach also has significant potential as a diagnostic tool for discrimination of cancer cells among shed urothelial cells in voided urine, and could lay the groundwork for an automated system for population screening for bladder cancer.

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Section: Discussionmentioning

confidence: 99%

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

Palmer

Litvinova

Rafailov

et al. 2015

Biomed. Opt. Express

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“…A shift of NADH from the free to the protein-bound state results from an increased contribution of glycolysis to energy production. 35 The elongated fluorescence lifetime τ 2 in diabetic subjects, especially in the short-wavelengths channel, is most likely interpretable as an increased contribution of free FAD. Field et al 12 published an increased fluorescence intensity of FAD in diabetic eyes.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy

et al. 2015

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Abstract. The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included in the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τ i , amplitudes α i , and relative contributions Q i were statistically compared between corresponding groups in two spectral channels (490 < ch1 < 560 nm, 560 < ch2 < 700 nm). The change in single fluorophores was estimated by applying the Holm-Bonferroni method and by calculating differences in the sum histograms of lifetimes. Median and mean of the histograms of τ 2 , τ 3 , and α 3 in ch1 show the greatest differences between phakic diabetic patients and age-matched controls (p < 0.000004). The lack of pixels with a τ 2 of ∼360 ps, the increased number of pixels with τ 2 > 450 ps, and the shift of τ 3 from ∼3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine dinucleotide at the fundus. AGE also accumulated in the crystalline lens.

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“…This long-range (1–10 nm) dipole-dipole coupling has emerged as a powerful technique to unravel the dynamics of biomolecules [ 25 ]. Numerous approaches to utilize FRET in protein studies have been developed over the past few years [ 65 , 66 , 67 , 68 , 69 ]. Several difficulties are encountered when using FRET in biological systems, for example the measured fluorescence intensities must be corrected for the auto-fluorescence of cells, and in most cases, the contribution from unbound fluorophores results in poor sensitivity, which presents difficulties in the interpretation of the results.…”

Section: Förster Resonance Energy Transfer (Fret)mentioning

confidence: 99%

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

Ghisaidoobe

Chung

2014

IJMS

725

474

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Förster resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (λEX ∼ 280 nm, λEM ∼ 350 nm), in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the protein’s) local environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic Förster resonance energy transfer (iFRET), a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins.

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Investigation of tryptophan–NADH interactions in live human cells using three-photon fluorescence lifetime imaging and Förster resonance energy transfer microscopy

Cited by 29 publications

References 9 publications

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

Detection of urinary bladder cancer cells using redox ratio and double excitation wavelengths autofluorescence

Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

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