NANO LETTERS
Photoactivation of Quantum Dot Fluorescence Following Endocytosis
2005 Vol. 5, No. 7 1445-1449
Jonathan Silver* and Wu Ou Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received April 29, 2005; Revised Manuscript Received May 23, 2005
ABSTRACT We studied the fluorescence of quantum dots in cells. Coating quantum dots with cationic peptides caused them to be endocytosed and transported to lysosomes. After overnight incubation, their fluorescence apparently dimmed but became markedly “photoactivatable”, increasing more than 3-fold within minutes on exposure to bright light, and decaying over hours in the dark. Photoactivation was greater in the presence of water than ethanol, and UV illumination compensated for lack of water during photoactivation. Dimming and photoactivation could affect the use of quantum dots as quantitative probes in vivo and lead to new uses, such as tracking molecular movement.
Quantum dots have great promise as fluorescent labels because they are brighter than traditional chemical fluors, resistant to photobleaching, tunable with respect to emission wavelength, and have narrow emission spectra, which allows multiplex labeling.1,2 Part of their promise in biology lies in being able to use them as probes in living cells and organisms.3,4 This goal will be advanced by better understanding of surface properties that target them to different biological structures5 and fluorescence properties in different environments. Certain positively charged peptides have been reported to cross cell membranes and promote the uptake of attached molecules6 including quantum dots.7 We confirmed the latter observation using streptavidin-coated, quantum dots from Quantum Dot Corporation (Hayward, CA) incubated with biotin-labeled poly-L-lysine. HeLa cells incubated overnight with 10nM quantum dots alone showed little fluorescence, whereas cells incubated with quantum dots mixed with 10 nM biotin-labeled poly-L-lysine contained bright fluorescent patches in the cytosol (Figure 1). It turned out that streptavidin and biotin were not required, as protein-A coated quantum dots mixed with nonbiotin-labeled poly-L-lysine (MW ∼ 100,000 kDa, Sigma Aldrich, St. Louis, MO) were taken up by cells equally well. Poly-L-lysine caused the quantum dots to aggregate in the absence of cells, indicated by the appearance of amorphous, fluorescent aggregates up to ∼2 µm in size by fluorescence microscopy. The CdSecore-ZnS-shell quantum dots have a polyacrylate coating, the negative charge of which helps prevent aggregation. It is likely that poly-L-lysine promoted aggregation of the quantum dots by binding electrostatically and cross-linking * Corresponding author: Building 4, Room 338, NIH, Bethesda, MD 20892, tel. (301) 496-3653, e-mail
[email protected].
them. Since the poly-L-lysine had about 100 lysine residues per molecule, it might also impart a net positive charge to bound quantum dots, which would promote binding to negatively charged sulfated glycosaminoglycans on the cell surface. When cells were examined in the first few hours after adding poly-L-lysine-quantum dot mixtures, quantum dot staining was largely on the cell surface (Figure 1a), whereas at later times most of the staining was intracellular (Figure 1b). Quantum Dot Corporation makes quantum dots that are further modified by attaching amino-poly(ethylene glycol) (PEG) to carboxyl groups in the acrylate coating. These dots did not aggregate and were not endocytosed when mixed with poly-L-lysine. However, streptavidin-coated versions of the PEG-modified quantum dots were endocytosed when mixed with a singly biotinylated, 9-mer arginine peptide8 (bio-arg9), giving a pattern in cells indistinguishable from that of non-PEGylated quantum dots mixed with poly-lysine. Extracellular aggregates were not observed in the case of PEG-modified, streptavidin quantum dots mixed with the bioarg9 peptide, indicating that aggregation was not required for endocytosis. Since detection of single quantum dots intracellularly has been reported,3 it was important to determine if the bright fluorescent spots we observed resulted from single quantum dots or aggregates. When we incubated cells with the bioarg9 peptide and a mixture of streptavidin quantum dots with emission maxima at 525 nm (green) and 655 nm (red), the intracellular fluorescent spots were mostly yellow (Figure 1c), whereas if cells were first incubated with peptide plus red-emitting quantum dots, followed by peptide plus greenemitting quantum dots, most of the intracellular fluorescent spots were either green or red (Figure 1d). This shows that
10.1021/nl050808n This article not subject to U.S. Copyright. Published 2005 by the American Chemical Society Published on Web 06/04/2005
Figure 1. Binding, uptake and aggregation of anionic quantum dots. HeLa cells were incubated with Qdots-655 plus poly-L-lysine for 4 h and then washed. Transmitted light and overlayed fluorescence images were taken after the wash (a) and 20 h later (b). Sequential versus simultaneous labeling with green and red fluorescent quantum dots. BHK cells were incubated overnight with bio-arg 9 peptide plus a mixture of streptavidin-coated Qdots with emission maxima at 525 and 655 nm (c), or with the peptide plus Qdots655 for 5 h and then the peptide plus Qdots525 overnight (d). Green and red fluorescence images are overlayed. Bar ) 20 µm.
Figure 2. Co-localization of intracellular quantum dots with late endosomes (LBPA) and lysosomes. (a-c) BHK cells incubated with streptavidin Qdots-655 plus bio-arg9 peptide overnight, then fixed (4% paraformaldehyde), permeabilized (0.1% Triton X-100), and stained with mouse monoclonal antibody 6C4 (from J. Gruenberg, University of Geneva) to LBPA and then with fluoresceinated anti-mouse IgG. (d-f) HeLa cells labeled with Qdots-655 plus poly-L-lysine overnight and then stained for 15 min with lysotracker green DND-26 (Molecular Probes, Eugene, OR). Bars ) 15 µm. Red fluorescence (a, d), green fluorescence (b, e), and overlaid, confocal fluorescence images (c, f) are shown with dual fluorescence in yellow. Arrows point to examples of co-localizing spots. 1446
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Figure 3. Photoactivation. HeLa cells labeled with poly-L-lysine plus streptavidin Qdots-655 were exposed to unattenuated 515-560 nm light from a 100 W mercury arc lamp with the diaphragm partially closed for 0 s (a), 45 s (b), or 90 s (c). The border of the central, photoactivated area is indicated by the white circle. After each time interval, the illumination intensity was turned to its minimum setting and a fluorescence image taken. Then the diaphragm was opened and the procedure repeated with bright illumination for an additional 0 s (d), 45 s (e), or 90 s (f). Bar ) 20 µm. g-h, 6 µm streptavidin-coated latex beads were incubated with biotinylated-Qdots655, washed to remove excess quantum dots, and added to HeLa cells that were labeled overnight with streptavidin-Qdots655 plus poly-L-lysine. Fluorescence images were taken without the confocal feature before and after 1 min illumination with 515-560 nm light from a 100 W mercury arc lamp. Yellow arrows point to intracellular quantum dots that brighten after illumination; white arrows point to quantum dotcoated latex beads, which show minimal brightening after illumination.
the intracellular spots we observed resulted from aggregates formed intracellularly, rather than single quantum dots. We estimated the size of intracellular quantum dot aggregates by comparing them to approximately equally bright, extracellular aggregates undergoing Brownian motion. The latter would stochastically leave a field of view in the microscope after a few seconds. From the depth of focus (∼10 µm) and the mean time to disappearance (∼10 s), we estimated the radius of such particles to be ∼200 nm.9 Spots of this size could contain more than 100 quantum dots, assuming quantum dot diameters, with coating, of 15 nm. The subcellular localization of quantum dot aggregates was investigated by staining with markers for endosomes and lysosomes. The quantum dot aggregates in cells labeled overnight partially co-localized with late endosome-multivesicular bodies, detected with monoclonal antibody to lysobisphosphatidic acid10 (LBPA) (Figure 2 a-c), and with lysosomes, stained with lysotracker dye (Figure 2 d-f). While most of the quantum dot aggregates in live cells were immobile, some of them moved, often in fairly straight Nano Lett., Vol. 5, No. 7, 2005
paths in a saltatory fashion. This motion was halted within 10 min of adding 5 µM nocodazole, a drug that destabilizes microtubules, implying that the aggregates move along tubulin tracks. The fluorescence intensity of intracellular quantum dot aggregates increased on exposure to bright light. To demonstrate this, the images in Figure 3 were taken with the mercury arc lamp intensity attenuated and the microscope diaphragm partially closed to illuminate the central portion of a field of view containing cells that had ingested polylysine-aggregated quantum dots. We turned up the light intensity for 15-second intervals, taking pictures under the same low light illumination after each interval. The fluorescence of the quantum dot aggregates increased over several intervals (Figure 3a-c). We then opened the diaphragm and continued the procedure. The fluorescence of quantum dot aggregates in the periphery then increased (Figure 3d-f). These results show that the fluorescence increase is extensive and not due to changes in the intensity of the exciting light in different pictures. By measuring average pixel intensity, we estimated that the fluorescence increased by at least 1447
Figure 4. Ethanol inhibition of photoactivation. HeLa cells were stained overnight with streptavidin-Qdots655 and poly-L-lysine, then fixed, permeabilized, rinsed, and covered with PBS (a,b) or ethanol (c-f). Fluorescence images were taken without confocal feature before (a, c, e) and after 1 min illumination with 515-560 nm light (b, d) or 340-380 nm light (f) from a 100 W mercury arc lamp and overlaid on transmitted light images. Bar ) 20 µm.
300%. We refer to this increase in fluorescence on exposure to light as “photoactivation”. Similar results with respect to photoactivation of endocytosed quantum dots were obtained with BHK cells and 293HEK cells, and with PEGylated streptavidin quantum dots mixed with bio-arg9 peptide. Photoactivation in our system required fairly high-intensity light. With the mercury arc lamp unattenuated and a 515560 nm band-pass excitation filter in the light path, the intensity of illuminating light as it exited the 63× objective was 1.6 mW, measured with a Newport model 1835-C MultiFunction optical meter. Photoactivation was less extensive using a 450-510 nm excitation filter, which produced 0.2 mW of light at the sample. Photoactivation was only weakly detectable using 488 and 565 nm laser sources in scanning mode, which provided 0.09 and 0.06 mW exiting the 63× lens. Photoactivation was much less intense in quantum dot aggregates formed in the absence of cells, e.g., quantum dots aggregated in solution with poly-L-lysine, or biotin-coated quantum dots adhered to streptavidin-coated latex beads. Figure 3g-h shows an experiment in which cells that had endocytosed quantum dots were mixed with 6 µm streptavidin-latex beads coated with biotin-quantum dots. The intracellular quantum dots (yellow arrows) photoactivated much more than the quantum dots coating the latex beads (white arrows). Consistent with enhancement of photoacti1448
vation by conditions inside the cell, little photoactivation was detected when quantum dots were predominantly on the surface of cells, 4 h after adding them to cells. Photoactivation has been reported in purely physical studies of quantum dots. In one study,11 heating quantum dots to 100° to 200° C caused progressive loss of fluorescence, postulated to be due to formation of surface defects that trapped electrons or holes, reducing their rate of recombination and hence photon emission. Bright light restored fluorescence to near starting levels. In another study of quantum dots dried on a surface,12 fluorescence increased over tens of seconds during bright illumination, then subsided over about 1 h in the absence of light, and required the continuous presence of water molecules to stay bright in the photoactivated state, suggesting that water molecules were involved in passivating surface defects. We observed similar time scales for photoactivation (∼1 min) and subsidence of enhanced fluorescence in cells kept in the dark (∼1 h). The time scale for photoactivation may depend on the intensity of the photoactivating light, which we did not investigate. To test for a requirement for water, we fixed and permeabilized cells that had taken up quantum dot aggregates and covered them with phosphate buffered saline (PBS) or ethanol. The quantum dots in PBS photoactivated more extensively than those in ethanol (Figure 4). Interestingly, when ultraviolet (UV) light was used for photoactivation, quantum dots in ethanol also photoactivated, Nano Lett., Vol. 5, No. 7, 2005
suggesting that the higher energy of UV photons or greater absorption of UV light by quantum dots compensates for reduced activity of ethanol versus water in photoactivation. Quantum dots are reported to undergo reversible darkening, termed “blinking”, possibly related to photoionization.13 Blinking is reportedly blocked by mercapto-ethanol;14 adding 140 mM 2-mercapto-ethanol to fixed, permeabilized cells that had taken up quantum dots did not markedly affect photoactivation in our system. We have shown that coating quantum dots with cationic peptides leads to their binding to cells, endocytosis, and transport to lysosomes. Similar findings have been reported with other positively charged proteins or peptides.15-18 Several hours after endocytosis, the fluorescence of endocytosed dots became markedly photoactivatable. It is likely that the photoactivation we observed occurs for quantum dots that become dim after endocytosis, since without photoactivation the fluorescence of intracellular quantum dot aggregates in lightly labeled cells was barely detectable (Figure 3), whereas after photoactivation it was approximately equal to that of endosome-size aggregates formed by poly-L-lysine ex vivo. We propose that a dark state is induced by interaction of quantum dots with cellular molecules, possibly involving degradation of the quantum dot surface in lysosomes. Surface coatings help maintain fluorescence properties of quantum dots and prevent release of cadmium selenide. Since cadmium selenide is toxic, degradation of the surface coating of quantum dots in lysosomes could affect their potential long-term toxicity.19 If degradation were extensive, it might result in a blue-shift of the emission peak due to a reduction in the semiconductor core diameter.13 We did not detect a blue-shift using a microscope that quantitated emitted light in 10 nm-wide bins, which suggests that under our conditions any degradation that occurred was mainly limited to the surface. The chemical events responsible for photoreversible, decreased fluorescence of quantum dots in certain conditions are not yet understood, but they may provide opportunities for new applications. Photoactivation was shown to be sensitive to CN- and could be used to measure submillimolar CN- concentration.20 If quantum dots can be photoreversibly dimmed by chemical or physical methods, or by conditions inside cells, photoactivation might be used to track the movement of cohorts of molecules labeled with quantum dots, as has been done with photoactivatable green
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fluorescent protein,21 by photoactivating a small region containing the molecules of interest. Acknowledgment. We thank Robert Bonner for help measuring light intensity, and Owen Schwartz, Jurai Kabat, and Michael Kruhlak for help with microscopy. References (1) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013-2016. (2) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (3) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538-544. (4) Gao, X.; Yang, L.; Petros, J. A.; Marshall, F. F.; Simons, J. W.; Nie, S. Curr. Opin. Biotechnol. 2005, 16, 63-72. (5) Hoshino, A.; Fujioka, K.; Oku, T.; Nakamura, S.; Suga, M.; Yamaguchi, Y.; Suzuki, K.; Yasuhara, M.; Yamamoto, K. Microbiol. Immunol. 2004, 48, 985-994. (6) Wadia, J.; Dowdy, S. F. AdV. Drug DeliV. ReV. 2005, 57, 579-596. (7) Gao, X. H.; Cui, Y. Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. M. Nat. Biotechnol. 2004, 22, 969-976. (8) Q-Tracker peptide, Quantum Dot Corporation. (9) We used the relations x2 ) 2D〈t〉 and D ) kT/6πηR, where x is half the depth of focus, 〈t〉 the mean time to disappearance, kT Boltzmann’s constant times the temperature, and η the fluid viscosity. (10) Matsuo, H.; Chevallier, J.; Mayran, N.; Le Blanc, I.; Ferguson, C.; Faure, J.; Blanc, N. S.; Matile, S.; Dubochet, J.; Sadoul, R.; Parton, R. G.; Vilbois, F.; Gruenberg, J. Science 2004, 303, 531-534. (11) Hess, B. C.; Okhrimenko, I. G.; Davis, R. C.; Stevens, B. C.; Schulzke, Q. A.; Wright, K. C.; Bass, C. D.; Evans, C. D.; Summers, S. L. Phys. ReV. Lett. 2001, 86, 3132-3135. (12) Cordero, S. R.; Carson, P. J.; Estabrook, R. A.; Strousse, G. F.; Buratto, S. K. J. Phys. Chem. B 2000, 1104, 12137-12142. (13) Van Sark, W. G. J. H. M.; Frederix, P. L. T. M.; Bol, A. A.; Gerritsen, H. C.; Meijerink, A. ChemPhysChem 2002, 3, 871-879. (14) Hohn, S.; Ha, T. J. Am. Chem. Soc. 2004, 126, 1324-1325. (15) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142-12150. (16) Mattheakis, L. C.; Dias, J. M.; Choi, Y.-J.; Gong, J.; Bruchez, M. P.; Liu, J.; Wang, E. Anal. Biochem. 2004, 327, 200-208. (17) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47-51. (18) Hanaki, K.; Momo, A.; Oku, T.; Komoto, A.; Maenosono, S.; Yamaguchi, Y.; Yamamoto, K. Biochem. Biophys. Res. Commun. 2003, 302, 496-501. (19) Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stolzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5, 331-338. (20) Jin, W. J.; Ferna´ndez-Argu¨elles, M. T.; Costa-Ferna´ndez, J. M.; Pereiro, R.; Sanz-Medel, A. Chem. Commun. (Cambridge) 2005, 7, 883-885. Epub Jan 04, 2005. (21) Patterson, G. H.; Lippincott-Schwartz, J. Science 2002, 297, 1873-1877.
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