Quantum Dot Applications to Neuroscience: New Tools for Probing Neurons and Glia

The Journal of Neuroscience, February 15, 2006 • 26(7):1893–1895 • 1893

Toolbox

Editor’s Note: Toolboxes are a new, occasional feature in the Journal designed to briefly highlight a new method or a resource of general use in neuroscience or to critically analyze existing approaches or methods. For more information, see http://www. jneurosci.org/misc/itoa.shtml.

Smita Pathak,1 Elizabeth Cao,2 Marie C. Davidson,3 Sungho Jin,1 and Gabriel A. Silva1,2,3,4 1Materials Science and Engineering Program, Departments of 2Bioengineering and 3Ophthalmology, and 4Neurosciences Program, University of California, San Diego, La Jolla, California 92037-0946 Key words: glia; immunocytochemistry; neurons; nanotechnology; quantum dots; imaging

Introduction though other work is pushing the devel- maekelbergh and Liljeroth, 2005). This Semiconductor fluorescent quantum dots opment of the technology forward. physical property represents another ma- are nanometer-sized functionalized parti- Quantum dots are nanometer-sized jor advantage over traditional organic flu- cles that display unique physical proper- particles composed of a heavy metal core, orophores that in general require distinct ties that make them particularly well such as cadmium selenium or cadmium chemistries to produce different colors. suited for visualizing and tracking molec- telluride with an intermediate unreactive For biological applications, quantum dots ular processes in cells using standard flu- zinc sulfide shell and a customized outer can be chemically functionalized to target orescence microscopy (Jaiswal et al., 2003; coating of different bioactive molecules proteins at high ligand-receptor densities. Watson et al., 2003; Michalet et al., 2005). tailored to a specific application (Fig. 1). Recent work has shown that, at least in They are readily excitable and have broad The composition and very small size of some cellular systems, quantum dots con- absorption spectra with very narrow quantum dots (5–8 nm) gives them jugated with natural ligands are readily in- emission spectra, allowing multiplexing unique and very stable fluorescent optical ternalized into cells, do not interfere with of many different colored quantum dots; properties that are readily tunable by intracellular signaling, and are nontoxic they display minimal photobleaching, changing their physical composition or (Chan et al., 2002; Murphy, 2002; Jain, thereby allowing molecular tracking over size. The photochemical properties of 2003; Watson et al., 2003; West and Halas, prolonged periods; they also display a quantum dots allow selective fluorescent 2003). blinking property that allows the identifi- tagging of proteins similar to classical im- cation of individual quantum dots. As a munocytochemistry. Additionally, the Quantum dots and neuroscience result, single molecule binding events can use of quantum dots is associated with Quantum dots represent a new tool of sig- be identified and tracked using optical flu- minimal photobleaching and a much nificant potential in neuroscience re- orescence microscopy, allowing the pur- higher signal-to-noise ratio. Their broad search. In addition to offering an alterna- suit of experiments that are difficult or not absorption spectra but very narrow emis- tive to traditional immunocytochemistry, possible given other experimental ap- sion spectra allows multiplexing of many they are particularly valuable for studies of proaches. Neuroscience-specific applica- quantum dots of different colors in the neurons and glia. Quantum dots can be tions of quantum dots are starting to same sample, something that cannot be used to visualize, measure, and track indi- emerge. Some work has focused on using achieved with traditional fluorophores. vidual molecular events using fluores- this nanotechnology to address cellular The physics responsible for these effects cence microscopy, and they provide the and molecular questions of interest, al- are beyond the scope of this brief intro- ability to visualize and track dynamic mo- duction, but the small size of quantum dot lecular processes over extended periods particles results in large but specific en- (e.g., from seconds to many minutes). ergy jumps between the energy band gaps These properties are difficult to achieve Received Sept. 11, 2005; revised Dec. 5, 2005; accepted Dec. 17, 2005. This work was supported by a Culpeper Biomedical Pilot Initiative of excited electron–hole pairs in the semi- using other techniques or approaches. For Award and the Stein Institute for Research on Aging. We thank Quantum conductor core. This effect results in example, quantum dots are useful for ex- Dot for providing the quantum dot kits used in our protocol. scaled changes of absorption and emis- periments that are limited by the re- Correspondence should be addressed to Dr. Gabriel A. Silva, University sion wavelengths as a function of particle stricted anatomy of neuronal and glial in- of California, San Diego, Jacobs Retina Center 0946, 9415 Campus Point Drive, La Jolla, CA 92037-0946. E-mail: [email protected]. size, so that small changes in the radius of teractions, such as the small size of the DOI:10.1523/JNEUROSCI.3847-05.2006 quantum dots translate into very distinct synaptic cleft, or between an astrocyte Copyright©2006SocietyforNeuroscience 0270-6474/06/261893-03$15.00/0 changes in color (Arya et al., 2005; Van- process and a neuron. Because of their ex- 1894 • J. Neurosci., February 15, 2006 • 26(7):1893–1895 Pathak et al. • Quantum Dot Labeling of Neurons and Glia tremely small size and optical resolution, they are also well suited for tracking the molecular dynamics of intracellular and/or intercellular molecular processes over long time scales. However, it should be appreciated that the hydrodynamic radius of functionalized quantum dots is larger (15–20 nm) than their actual size of 5–8 nm (Larson et al., 2003). Recent studies using quantum dots in neuroscience illustrate the potential of this technology. Triller and colleagues (Dahan et al., 2003) used antibody functionalized quantum Figure 1. Structure of a semiconductor fluorescent quantum dot nanocrystal. The heavy metal core is responsible for the dots to track the lateral diffusion of gly- fluorescence properties of the quantum dot. The nonemissive shell stabilizes the core, whereas the coating layer provides anchor cine receptors in cultures of primary spi- sites to organic and biological ligands such as antibodies, peptides, and other organic molecules. nal cord neurons. They were able to track the trajectory of individual glycine receptors for tens of minutes at spatial resolu- tions of 5–10 nm, demonstrating that the diffusion dynamics varied depending on whether the receptors were synaptic, peri- synaptic, or extrasynaptic. Vu et al. (2005) tagged nerve growth factor (␤NGF) to quantum dots and used them to promote neuronal-like differentiation in cultured pheochromocytoma 12 (PC12) cells. Ulti- mately, these approaches could be used to visualize and track functional responses in neurons. However, as with any new technology, there are caveats. For example, Vu et al. (2005) reported that ␤NGF conjugated to quantum dots had reduced activ- ity compared with free ␤NGF. Other groups are pushing the technology forward and providing new quantum-dot- based tools. Brinker and colleagues (Fan et al., 2005) developed a technique to produce biocompatible water-soluble quantum dot micelles that retain the optical properties of individual quantum dots. These micelles showed uptake and intracellular dispersion in cultured hippo- Figure 2. Fluorescent labeling of neurons and glia with antibody-conjugated 605 nm quantum dots. A, Primary cortical campal neurons. Ting and colleagues neurons specifically labeled for ␤-tubulin. B, C, Primary cortical astrocytes specifically labeled for glial fibrillary acidic protein (Howarth et al., 2005) are developing a (GFAP). D, F, PC12 cells labeled for ␤-tubulin. E, r-MC1 neural retinal Muller glial cells specifically labeled for GFAP. G, PC12 cells modified quantum dot labeling approach labeled for ␤-tubulin using standard immunocytochemistry. H, Primary spinal cord astrocytes labeled for GFAP using standard that addresses the relatively large size of immunocytochemistry. I, An example of artifactual nonspecific labeling in r-MC1 Muller cells with anti-GFAP-conjugated 605 nm antibody–quantum-dot conjugates and quantum dots. In this case, putative nonspecific electrostatic interactions between quantum dots and cellular proteins led to the instability of some quantum-dot–li- intense nuclear staining and mild cytoplasmic staining using other quantum dot conjugation protocols described for mammalian gand interactions. Their technique tags cells.Allimagingparameterswereconstantforthedifferentexperimentalconditions,withanacquisition/exposuretimeof30ms for all panels, except for I, which was taken with an acquisition time of 100 ms. cell surface proteins with a specific peptide (a 15 aa polypeptide called acceptor neural mammalian cells in the peer- of quantum dots in glial cells. Given the protein; GLNDIFEAQKIEVWHE) that unique properties that quantum dots have can be directly biotinylated as a target for reviewed literature and in instructions to offer neuroscience research, their fu- streptavidin-conjugated quantum dots. from commercial sources do not always ture looks pretty bright (and stable). Using this approach, they were able to readily work with primary neurons, glia, specifically label and track AMPA recep- and related cell lines (Fig. 2I). In the sup- References tors on cultured hippocampal neurons. plemental data, we present a very simple Arya H, Kaul Z, Wadhwa R, Taira K, Hirano T, Ultimately, quantum dot nanotech- and straightforward protocol for specific Kaul SC (2005) Quantum dots in bio-imag- nologies will require easy-to-use ap- labeling of both neurons and glia (Fig. 2 ing: revolution by the small. Biochem Biophys proaches that can be straightforwardly and supplemental Table 1, available at Res Commun 329:1173–1177. Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, replicated in a typical neurobiology lab. www.jneurosci.org as supplemental ma- Nie S (2002) Luminescent quantum dots for Unfortunately, in our experience, most terial). Of particular note, as far as we are multiplexed biological detection and imaging. quantum dot protocols intended for non- aware, these results represent the first use Curr Opin Biotechnol 13:40–46. Pathak et al. • Quantum Dot Labeling of Neurons and Glia J. Neurosci., February 15, 2006 • 26(7):1893–1895 • 1895

Dahan M, Levi S, Luccardini C, Rostaing P, Jaiswal JK, Mattoussi H, Mauro JM, Simon SM Vanmaekelbergh D, Liljeroth P (2005) Electron- Riveau B, Triller A (2003) Diffusion dynam- (2003) Long-term multiple color imaging of conducting quantum dot solids: novel materi- ics of glycine receptors revealed by single- live cells using quantum dot bioconjugates. als based on colloidal semiconductor nanoc- quantum dot tracking. Science 302:442–445. Nat Biotechnol 21:47–51. rystals. Chem Soc Rev 34:299–312. Fan H, Leve EW, Scullin C, Gabaldon J, Tallant D, Larson DR, Zipfel WR, Williams RM, Clark SW, Vu TQ, Maddipati R, Blute TA, Nehilla BJ, Nusb- Bunge S, Boyle T, Wilson MC, Brinker CJ Bruchez MP, Wise FW, Webb WW (2003) lat L, Desai TA (2005) Peptide-conjugated (2005) Surfactant-assisted synthesis of water- Water-soluble quantum dots for multiphoton quantum dots activate neuronal receptors and soluble and biocompatible semiconductor fluorescence imaging in vivo. Science initiate downstream signaling of neurite quantum dot micelles. Nano Lett 5:645–648. 300:1434–1436. growth. Nano Lett 5:603–607. Howarth M, Takao K, Hayashi Y, Ting AY (2005) Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Watson A, Wu X, Bruchez M (2003) Lighting up Targeting quantum dots to surface proteins in Doose S, Li JJ, Sundaresan G, Wu AM, Gam- cells with quantum dots. Biotechniques living cells with biotin ligase. Proc Natl Acad bhir SS, Weiss S (2005) Quantum dots for 34:296–300, 302–303. Sci USA 102:7583–7588. live cells, in vivo imaging, and diagnostics. Sci- West JL, Halas NJ (2003) Engineered nanoma- Jain KK (2003) Nanodiagnostics: application of ence 307:538–544. terials for biophotonics applications: improv- nanotechnology in molecular diagnostics. Ex- Murphy CJ (2002) Optical sensing with quan- ing sensing, imaging, and therapeutics. Annu pert Rev Mol Diagn 3:153–161. tum dots. Anal Chem 74:520A–526A. Rev Biomed Eng 5:285–292.