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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.

Show MeSH
Fluorescent tracking of QD uptake and breakdown in cells (cellular structures not to scale). (a) QDs conjugated to an electron donor can bind to specific receptors (gray sphere) but are not immediately fluorescent.  QDs that have lost this surface cap are immediately green-fluorescent (green spheres) but do not bind to receptors and are rarely endocytosed.  (b) Processing through the cell leads to changes in surface cap and resulting alterations of fluorescence.  Areas of normal cellular redox potential such as endosomes (gray) are not fluorescent unless illuminated for significant amounts of time.  Highly oxidizing areas such as lysosomes, in contrast, show immediate green fluorescence and no blue fluorescence without photoenhancement, indicating that the conjugate has been removed from the particle, probably due to proteases in the lysosome.  Particles near mitochondria show varying degrees of green and blue fluorescence with slow photoenhancement.
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fig8: Fluorescent tracking of QD uptake and breakdown in cells (cellular structures not to scale). (a) QDs conjugated to an electron donor can bind to specific receptors (gray sphere) but are not immediately fluorescent. QDs that have lost this surface cap are immediately green-fluorescent (green spheres) but do not bind to receptors and are rarely endocytosed. (b) Processing through the cell leads to changes in surface cap and resulting alterations of fluorescence. Areas of normal cellular redox potential such as endosomes (gray) are not fluorescent unless illuminated for significant amounts of time. Highly oxidizing areas such as lysosomes, in contrast, show immediate green fluorescence and no blue fluorescence without photoenhancement, indicating that the conjugate has been removed from the particle, probably due to proteases in the lysosome. Particles near mitochondria show varying degrees of green and blue fluorescence with slow photoenhancement.

Mentions: In cells, a minimum number of dopamines, corresponding to slow enhancement, is necessaryfor uptake. However, as the particlestravel through the cell, particularly to oxidizing regions, enhancement becomesmore rapid suggesting that the cap decay mechanism is in fact correct. Thesedata could be used for a semiquantitative model of QD processing in cells(Schematic in Figure 8).


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)

Fluorescent tracking of QD uptake and breakdown in cells (cellular structures not to scale). (a) QDs conjugated to an electron donor can bind to specific receptors (gray sphere) but are not immediately fluorescent.  QDs that have lost this surface cap are immediately green-fluorescent (green spheres) but do not bind to receptors and are rarely endocytosed.  (b) Processing through the cell leads to changes in surface cap and resulting alterations of fluorescence.  Areas of normal cellular redox potential such as endosomes (gray) are not fluorescent unless illuminated for significant amounts of time.  Highly oxidizing areas such as lysosomes, in contrast, show immediate green fluorescence and no blue fluorescence without photoenhancement, indicating that the conjugate has been removed from the particle, probably due to proteases in the lysosome.  Particles near mitochondria show varying degrees of green and blue fluorescence with slow photoenhancement.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Fluorescent tracking of QD uptake and breakdown in cells (cellular structures not to scale). (a) QDs conjugated to an electron donor can bind to specific receptors (gray sphere) but are not immediately fluorescent. QDs that have lost this surface cap are immediately green-fluorescent (green spheres) but do not bind to receptors and are rarely endocytosed. (b) Processing through the cell leads to changes in surface cap and resulting alterations of fluorescence. Areas of normal cellular redox potential such as endosomes (gray) are not fluorescent unless illuminated for significant amounts of time. Highly oxidizing areas such as lysosomes, in contrast, show immediate green fluorescence and no blue fluorescence without photoenhancement, indicating that the conjugate has been removed from the particle, probably due to proteases in the lysosome. Particles near mitochondria show varying degrees of green and blue fluorescence with slow photoenhancement.
Mentions: In cells, a minimum number of dopamines, corresponding to slow enhancement, is necessaryfor uptake. However, as the particlestravel through the cell, particularly to oxidizing regions, enhancement becomesmore rapid suggesting that the cap decay mechanism is in fact correct. Thesedata could be used for a semiquantitative model of QD processing in cells(Schematic in Figure 8).

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