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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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Related in: MedlinePlus

Confocal images and time courses of QD-dopamine with 100 ± 10 DA/particle in PC12-dopamine receptor cells colabeled with organelle dyes. Scale bar = 10 μm for all panels; in all panels, the green channel indicates the QDs and the red channel the organelle dye. The white lines indicate the cell nuclei.  (a) Paraformaldehyde-fixed cell labeled with MitoTracker and QDs.  (b) The same cell after 30 seconds of laser-light exposure. (c) Live cells labeled with MitoTracker and QDs.  (d) The same cells after 30 seconds of laser-light exposure.  (e) Live cell labeled with LysoTracker and QDs.  (f) The same cell after 30 seconds of light exposure. (g) Relative intensities from the indicated regions over 100 seconds of exposure time.
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fig4: Confocal images and time courses of QD-dopamine with 100 ± 10 DA/particle in PC12-dopamine receptor cells colabeled with organelle dyes. Scale bar = 10 μm for all panels; in all panels, the green channel indicates the QDs and the red channel the organelle dye. The white lines indicate the cell nuclei. (a) Paraformaldehyde-fixed cell labeled with MitoTracker and QDs. (b) The same cell after 30 seconds of laser-light exposure. (c) Live cells labeled with MitoTracker and QDs. (d) The same cells after 30 seconds of laser-light exposure. (e) Live cell labeled with LysoTracker and QDs. (f) The same cell after 30 seconds of light exposure. (g) Relative intensities from the indicated regions over 100 seconds of exposure time.

Mentions: The locationand appearance of various organelles in cells were determined bylabeling with specific dyes such as Lysotracker and Mitotracker (see Figure 4). Studies of unquenching time-courses of thesimultaneously loaded QD-dopamine were then performed under 488 nm laserillumination. The results showed aconsistent and reproducible pattern with three distinctive QD behaviors. Infixed cells, with depolarized mitochondria, QDs did not show overlap with themitochondrial-targeting dye MitoTracker.QD fluorescence brightened slightly under light exposure (see Figures 4(a),4(b), and 4(g)). In live cells, however, themitochondrial region brightened quickly and intensely, with QD-Mitotrackeroverlap becoming apparent (see Figures 4(c),4(d), and 4(g)). In both live andfixed cells, a good deal of QD fluorescence was seen colocalized withlysosomes. QD fluorescence withinlysosomes shows only bleaching with time (see Figures 4(e),4(f), and 4(g)). QDsthat were outside the cell, in aggregates outside the membrane, exhibited nobrightening but only photobleaching over time (see Figures 4(e),4(f), and 4(g)).


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)

Confocal images and time courses of QD-dopamine with 100 ± 10 DA/particle in PC12-dopamine receptor cells colabeled with organelle dyes. Scale bar = 10 μm for all panels; in all panels, the green channel indicates the QDs and the red channel the organelle dye. The white lines indicate the cell nuclei.  (a) Paraformaldehyde-fixed cell labeled with MitoTracker and QDs.  (b) The same cell after 30 seconds of laser-light exposure. (c) Live cells labeled with MitoTracker and QDs.  (d) The same cells after 30 seconds of laser-light exposure.  (e) Live cell labeled with LysoTracker and QDs.  (f) The same cell after 30 seconds of light exposure. (g) Relative intensities from the indicated regions over 100 seconds of exposure time.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Confocal images and time courses of QD-dopamine with 100 ± 10 DA/particle in PC12-dopamine receptor cells colabeled with organelle dyes. Scale bar = 10 μm for all panels; in all panels, the green channel indicates the QDs and the red channel the organelle dye. The white lines indicate the cell nuclei. (a) Paraformaldehyde-fixed cell labeled with MitoTracker and QDs. (b) The same cell after 30 seconds of laser-light exposure. (c) Live cells labeled with MitoTracker and QDs. (d) The same cells after 30 seconds of laser-light exposure. (e) Live cell labeled with LysoTracker and QDs. (f) The same cell after 30 seconds of light exposure. (g) Relative intensities from the indicated regions over 100 seconds of exposure time.
Mentions: The locationand appearance of various organelles in cells were determined bylabeling with specific dyes such as Lysotracker and Mitotracker (see Figure 4). Studies of unquenching time-courses of thesimultaneously loaded QD-dopamine were then performed under 488 nm laserillumination. The results showed aconsistent and reproducible pattern with three distinctive QD behaviors. Infixed cells, with depolarized mitochondria, QDs did not show overlap with themitochondrial-targeting dye MitoTracker.QD fluorescence brightened slightly under light exposure (see Figures 4(a),4(b), and 4(g)). In live cells, however, themitochondrial region brightened quickly and intensely, with QD-Mitotrackeroverlap becoming apparent (see Figures 4(c),4(d), and 4(g)). In both live andfixed cells, a good deal of QD fluorescence was seen colocalized withlysosomes. QD fluorescence withinlysosomes shows only bleaching with time (see Figures 4(e),4(f), and 4(g)). QDsthat were outside the cell, in aggregates outside the membrane, exhibited nobrightening but only photobleaching over time (see Figures 4(e),4(f), and 4(g)).

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