Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

Földes-Papp, Zeno; Liao, Shih-Chu Jeff; You, Tiefeng; Barbieri, Beniamino

doi:10.2174/138920109788922137

Cited by 8 publications

(3 citation statements)

References 33 publications

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“…A second source of fluctuations that is particularly relevant at low light levels or short integration times, is the variation of the photon counts due to the stochastic nature of light emission and detection [18–20]. Other fluctuation sources can be fluorescence background, impurities or scattered light [21–23]. …”

Section: Introductionmentioning

confidence: 99%

Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments

Turgeman

Fixler

2013

Biomed. Opt. Express

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In fluorescence fluctuation polarization sensitive experiments, the limitations associated with detecting the rotational timescale are usually eliminated by applying fluorescence correlation spectroscopy analysis. In this paper, the variance of the time-averaged fluorescence intensity extracted from the second moment of the measured fluorescence intensity is analyzed in the short time limit, before fluctuations resulting from rotational diffusion average out. Since rotational correlation times of fluorescence molecules are typically much lower than the temporal resolution of the system, independently of the time bins used, averaging over an ensemble of time-averaged trajectories was performed in order to construct the time-averaged intensity distribution, thus improving the signal-to-noise ratio. Rotational correlation times of fluorescein molecules in different viscosities of the medium within the range of the anti-bunching time (1-10 ns) were then extracted using this method.

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Section: Introductionmentioning

confidence: 99%

Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments

Turgeman

Fixler

2013

Biomed. Opt. Express

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“…6,7 In the past decade, strategies for reducing fluorescence backgrounds in microfluidic systems have been extensively studied to accommodate the increasing applications of these systems in biological and medical tests. Some examples of these strategies are: covering the channel surface with hydrophilic or blocking reagents, [8][9][10][11][12][13] fabricating microchannels with substrates having lower hydrophobicity, 14,15 and using innovative optical arrangements 16,17 or mathematical algorithms 18 to filter out background noise. The first two methods prevent the nonspecific absorption of fluorescent molecules onto a surface; however, they are not able to decrease the background fluorescence resulting from the dye molecules in the solution.…”

Section: Introductionmentioning

confidence: 99%

Enhanced fluorescence detection using liquid–liquid extraction in a microfluidic droplet system

Chen

Wang

2012

Lab Chip

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Reducing the fluorescence background in microfluidic assays is important in obtaining accurate outcomes and enhancing the quality of detections. This study demonstrates an integrated process including cell labelling, fluorescence background reduction, and biomolecule detection using liquid-liquid extraction in a microfluidic droplet system. The cellular lipids in Chlorella vulgaris and NIH/3T3 cells were labelled with a hydrophobic dye, Nile red, to investigate the performance of the proposed method. The fluorescence background of the lipid detection can be reduced by 85% and the removal efficiency increased with the volume of continuous phase surrounding a droplet. The removal rate of the fluorescence background increased as the surface area to volume ratio of a droplet increased. Before Nile red was removed from the droplet, the signal to noise ratio was as low as 1.30 and it was difficult to distinguish cells from the background. Removing Nile red increased the signal to noise ratio to 22 and 34 for Chlorella vulgaris and NIH/3T3, respectively, and these were 17 fold and 10 fold of the values before extraction. The proposed method successfully demonstrates the enhancement of fluorescence detection of cellular lipids and has great potential in improving other fluorescence-based detections in microfluidic systems.

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“…Furthermore, the advent of fluorescence lifetime microscopy (FLIM) provides the tools for extrapolating the advantages of TRPS to microscopy. 17 Some recent manuscripts have highlighted the importance of using time-gating methods to enhance the signal resolution in applications such as single-molecule fluorescence spectroscopy, 18 fluorescence correlation spectroscopy, 19 and luminescence microscopy. 20 Notwithstanding, there is a lack of a general methodology on how to optimally use TRPS to improve S/B of photoluminescent probes.…”

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confidence: 99%

Optimizing the Sensitivity of Photoluminescent Probes Using Time-Resolved Spectroscopy: A Molecular Beacon Case Study

Huang

Martí

2012

Anal. Chem.

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Improving probes so that they can perform more sensitive and accurate detections is at the heart of much fundamental and applied research. Within the past few years a considerable amount of effort has been devoted to the study of photoluminescent probes in combination with time-resolved photoluminescence spectroscopy (TRPS). Although TRPS is a powerful and important technique for improving the sensitivity of long-lived probes, there is a lack of a general methodology that would allow one to unambiguously optimize the parameter affecting this technique. In this manuscript it will be shown how parameters that are probe- and technique-specific can affect the effectiveness of TRPS in improving sensitivity. Furthermore, it will be demonstrated that, when TRPS is used, the sensitivity of the probe is strongly dependent on the time window used to generate the time-resolved emission spectra (TRES). A method will be described that will allow one to remove the uncertainty in the selection of the time window that would yield the optimum improvement in probe performance, as well as the experimental parameters that need to be considered. Molecular beacon probes (MBs) were used to demonstrate these points. These probes show signal-to-background ratios (S/B) of less than 9 when SSPS is used, which can be easily enhanced to 17 using TRPS. The detection limits were also improved when TRPS is used allowing detecting target DNA with concentrations as low as 13.6 nM.

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Reducing Background Contributions in Fluorescence Fluctuation Time- Traces for Single-Molecule Measurements in Solution

Cited by 8 publications

References 33 publications

Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments

Time-averaged fluorescence intensity analysis in fluorescence fluctuation polarization sensitive experiments

Enhanced fluorescence detection using liquid–liquid extraction in a microfluidic droplet system

Optimizing the Sensitivity of Photoluminescent Probes Using Time-Resolved Spectroscopy: A Molecular Beacon Case Study

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