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Fluorescence intensity and intermittency as tools for following dopamine bioconjugate processing in living cells.

Khatchadourian R, Bachir A, Clarke SJ, Heyes CD, Wiseman PW, Nadeau JL - J. Biomed. Biotechnol. (2007)

Bottom Line: CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching.In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells.This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, McGill University, 3775 Rue University, 316 Lyman Duff Medical Building, Montréal, Canada.

ABSTRACT
CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching. In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells. This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

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Evaluation of QD blinking.  (a) Typical image of immobilized QDs. Due to their different brightness, some spots appear larger than others. Single QDs are identified by the size of their point spread function and selected for subsequent analysis. Image size is 40 × 40 μm2. (b) Typical intensity trace of a single QD under continuous excitation. The grey line indicates an arbitrary threshold used to separate on-events from off-events. The threshold is usually set to 2–3 standard deviations above the background noise level, which is determined from nearby pixels containing no QD.
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fig6: Evaluation of QD blinking. (a) Typical image of immobilized QDs. Due to their different brightness, some spots appear larger than others. Single QDs are identified by the size of their point spread function and selected for subsequent analysis. Image size is 40 × 40 μm2. (b) Typical intensity trace of a single QD under continuous excitation. The grey line indicates an arbitrary threshold used to separate on-events from off-events. The threshold is usually set to 2–3 standard deviations above the background noise level, which is determined from nearby pixels containing no QD.

Mentions: Blinkingis conveniently studied by taking an image series with time of a number ofimmobilized QDs [9–11].Details of the analysis may be found in the methods section and in previous publications [9, 10]. The result of the analysis is that the fluorescence time trace for each identified QD isextracted from the image series, and the durations of on and off times (time durations forwhich the signal is above and below the threshold level, resp.) areextracted. A typical image and extracted fluorescence trace of a single,immobile QD are shown in Figure 6.


Fluorescence intensity and intermittency as tools for following dopamine bioconjugate processing in living cells.

Khatchadourian R, Bachir A, Clarke SJ, Heyes CD, Wiseman PW, Nadeau JL - J. Biomed. Biotechnol. (2007)

Evaluation of QD blinking.  (a) Typical image of immobilized QDs. Due to their different brightness, some spots appear larger than others. Single QDs are identified by the size of their point spread function and selected for subsequent analysis. Image size is 40 × 40 μm2. (b) Typical intensity trace of a single QD under continuous excitation. The grey line indicates an arbitrary threshold used to separate on-events from off-events. The threshold is usually set to 2–3 standard deviations above the background noise level, which is determined from nearby pixels containing no QD.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279208&req=5

fig6: Evaluation of QD blinking. (a) Typical image of immobilized QDs. Due to their different brightness, some spots appear larger than others. Single QDs are identified by the size of their point spread function and selected for subsequent analysis. Image size is 40 × 40 μm2. (b) Typical intensity trace of a single QD under continuous excitation. The grey line indicates an arbitrary threshold used to separate on-events from off-events. The threshold is usually set to 2–3 standard deviations above the background noise level, which is determined from nearby pixels containing no QD.
Mentions: Blinkingis conveniently studied by taking an image series with time of a number ofimmobilized QDs [9–11].Details of the analysis may be found in the methods section and in previous publications [9, 10]. The result of the analysis is that the fluorescence time trace for each identified QD isextracted from the image series, and the durations of on and off times (time durations forwhich the signal is above and below the threshold level, resp.) areextracted. A typical image and extracted fluorescence trace of a single,immobile QD are shown in Figure 6.

Bottom Line: CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching.In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells.This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, McGill University, 3775 Rue University, 316 Lyman Duff Medical Building, Montréal, Canada.

ABSTRACT
CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching. In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells. This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions.

Show MeSH
Related in: MedlinePlus