Abstract
For decades, optical studies of single molecules have relied on fluorescence detection. The availability of alternative approaches for single-molecule interrogation would greatly expand the range of addressable molecules beyond species that are highly emissive and photostable, thus offering new applications in fields other than molecular biophysics and imaging. Here, we discuss the range of recent developments in optical label-free detection and imaging schemes that offer single-molecule sensitivity, with an emphasis on plasmonically enhanced and scattering-based approaches. We highlight the advantages and challenges facing these emerging methodologies and briefly outline their potential future applications.
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References
Lounis, B. & Orrit, M. Single-photon sources. Rep. Prog. Phys. 68, 1129–1179 (2005).
Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).
Maxwell, J. C. Molecules. Nature 8, 437–441 (1873).
Mamin, H. J. et al. Nanoscale nuclear magnetic resonance with a nitrogen-vacancy spin sensor. Science 339, 557–560 (2013).
Staudacher, T. et al. Nuclear magnetic resonance spectroscopy on a (5-nanometer)3 sample volume. Science 339, 561–563 (2013).
McKeever, J. et al. Deterministic generation of single photons from one atom trapped in a cavity. Science 303, 1992–1994 (2004).
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Harris, T. D. et al. Single-molecule DNA sequencing of a viral genome. Science 320, 106–109 (2008).
Santori, C., Fattal, D., Vučković, J., Solomon, G. S. & Yamamoto, Y. Indistinguishable photons from a single-photon device. Nature 419, 594–597 (2002).
Moerner, W. E. & Kador, L. Optical detection and spectroscopy of single molecules in a solid. Phys. Rev. Lett. 62, 2535–2538 (1989).
Orrit, M. & Bernard, J. Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys. Rev. Lett. 65, 2716–2719 (1990).
Hwang, J. et al. A single-molecule optical transistor. Nature 460, 76–80 (2009).
Kukura, P., Celebrano, M., Renn, A. & Sandoghdar, V. Single-molecule sensitivity in optical absorption at room temperature. J. Phys. Chem. Lett. 1, 3323–3327 (2010).
Kaiser, H. J. et al. Order of lipid phases in model and plasma membranes. Proc. Natl Acad. Sci. USA 106, 16645–16650 (2009).
Staneva, G., Seigneuret, M., Conjeaud, H., Puff, N. & Angelova, M. I. Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1. Langmuir 27, 15074–15082 (2011).
Mascalchi, P., Haanappel, E., Carayon, K., Mazères, S. & Salomé, L. Probing the influence of the particle in single particle tracking measurements of lipid diffusion. Soft Matter 8, 4462–4470 (2012).
Rajapaksa, I. & Wickramasinghe, H. K. Raman spectroscopy and microscopy based on mechanical force detection. Appl. Phys. Lett. 99, 161103 (2011).
Chen, C., Chu, P., Bobisch, C. A., Mills, D. L. & Ho, W. Viewing the interior of a single molecule: vibronically resolved photon imaging at submolecular resolution. Phys. Rev. Lett. 105, 217402 (2010).
Hwang, J., Fejer, M. & Moerner, W. E. Scanning interferometric microscopy for the detection of ultrasmall phase shifts in condensed matter. Phys. Rev. A 73, 021802 (2006).
Hofkens, J. & Roeffaers, M. B. J. Microscopy: single-molecule light absorption. Nature Photon. 5, 80–81 (2011).
Celebrano, M., Kukura, P., Renn, A. & Sandoghdar, V. Single-molecule imaging by optical absorption. Nature Photon. 5, 95–98 (2011).
Chong, S., Min, W. & Xie, X. S. Ground-state depletion microscopy: detection sensitivity of single-molecule optical absorption at room temperature. J. Phys. Chem. Lett. 1, 3316–3322 (2010).
Gaiduk, A., Yorulmaz, M., Ruijgrok, P. V. & Orrit, M. Room-temperature detection of a single molecule's absorption by photothermal contrast. Science 330, 353–356 (2010).
Fleischmann, M., Hendra, P. J. & McQuillan, A. J. Raman spectra of pyridine adsorbed at a silver electrode. Chem. Phys. Lett. 26, 163–166 (1974).
Jeanmaire, D. L. & Van Duyne, R. P. Surface Raman spectroelectrochemistry. J. Electronanal. Chem. 84, 1–20 (1977).
Nie, S. & Emory, S. R. Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 275, 1102–1106 (1997).
Yampolsky, S. et al. Seeing a single molecule vibrate through time-resolved coherent anti-Stokes Raman scattering. Nature Photon. 8, 650–656 (2014).
Stöckle, R. M., Suh, Y. D., Deckert, V. & Zenobi, R. Nanoscale chemical analysis by tip-enhanced Raman spectroscopy. Chem. Phys. Lett. 318, 131–136 (2000).
Wang, Y. & Irudayaraj, J. Surface-enhanced Raman spectroscopy at single-molecule scale and its implications in biology. Phil. Trans. R. Soc. Lond. B 368, 20120026 (2012).
Sonntag, M. D. et al. Single-molecule tip-enhanced Raman spectroscopy. J. Phys. Chem. C 116, 478–483 (2011).
Zhang, R. et al. Chemical mapping of a single molecule by plasmon-enhanced Raman scattering. Nature 498, 82–86 (2013).
Thacker, V. V. et al. DNA origami based assembly of gold nanoparticle dimers for surface-enhanced Raman scattering. Nature Commun. 5, 3448 (2014).
Galloway, C. M. et al. Plasmon-assisted delivery of single nano-objects in an optical hot spot. Nano Lett. 13, 4299–4304 (2013).
Ament, I., Prasad, J., Henkel, A., Schmachtel, S. & Sönnichsen, C. Single unlabeled protein detection on individual plasmonic nanoparticles. Nano Lett. 12, 1092–1095 (2012).
Zijlstra, P., Paulo, P. M. R. & Orrit, M. Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod. Nature Nanotech. 7, 379–382 (2012).
Beuwer, M. A., Prins, M. W. J. & Zijlstra, P. Stochastic protein interactions monitored by hundreds of single-molecule plasmonic biosensors. Nano Lett. 15, 3507–3511 (2015).
Al Balushi, A. A., Zehtabi-Oskuie, A. & Gordon, R. Observing single protein binding by optical transmission through a double nanohole aperture in a metal film. Biomed. Opt. Express 4, 1504–1511 (2013).
Wheaton, S., Gelfand, R. M. & Gordon, R. Probing the Raman-active acoustic vibrations of nanoparticles with extraordinary spectral resolution. Nature Photon. 9, 68–72 (2014).
Weigel, A. & Kukura, P. Acoustic vibrational spectroscopy: Raman extraordinaire. Nature Photon. 9, 11–12 (2014).
Vollmer, F. & Arnold, S. Whispering-gallery-mode biosensing: label-free detection down to single molecules. Nature Methods 5, 591–596 (2008).
Dantham, V. R. et al. Label-free detection of single protein using a nanoplasmonic-photonic hybrid microcavity. Nano Lett. 13, 3347–3351 (2013).
Baaske, M. D., Foreman, M. R. & Vollmer, F. Single-molecule nucleic acid interactions monitored on a label-free microcavity biosensor platform. Nature Nanotech. 9, 933–939 (2014).
Armani, A. M., Kulkarni, R. P., Fraser, S. E., Flagan, R. C. & Vahala, K. J. Label-free, single-molecule detection with optical microcavities. Science 317, 783–787 (2007).
Andrecka, J., Spillane, K. M., Ortega Arroyo, J. & Kukura, P. Direct observation and control of supported lipid bilayer formation with interferometric scattering microscopy. ACS Nano 7, 10662–10670 (2013).
de Wit, G., Danial, J. S. H., Kukura, P. & Wallace, M. I. Dynamic label-free imaging of lipid nanodomains. Proc. Natl Acad. Sci. USA 112, 12299–12303 (2015).
Cohen, S. I. A. et al. A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers. Nature Struct. Mol. Biol. 22, 207–213 (2015).
Dumont, E. L. P., Do, C. & Hess, H. F. Molecular wear of microtubules propelled by surface-adhered kinesins. Nature Nanotech. 10, 166–169 (2015).
Lindfors, K., Kalkbrenner, T., Stoller, P. & Sandoghdar, V. Detection and spectroscopy of gold nanoparticles using supercontinuum white light confocal microscopy. Phys. Rev. Lett. 93, 037401 (2004).
Kukura, P. et al. High-speed nanoscopic tracking of the position and orientation of a single virus. Nature Methods 6, 923–927 (2009).
Ortega Arroyo, J. & Kukura, P. Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy. Phys. Chem. Chem. Phys. 14, 15625–15636 (2012).
Ortega Arroyo, J. et al. Label-free, all-optical detection, imaging, and tracking of a single protein. Nano Lett. 14, 2065–2070 (2014).
Piliarik, M. & Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites. Nature Commun. 5, 4495 (2014).
Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).
Sonntag, M. D., Chulhai, D., Seideman, T., Jensen, L. & Van Duyne, R. P. The origin of relative intensity fluctuations in single-molecule tip-enhanced Raman spectroscopy. J. Am. Chem. Soc. 135, 17187–17192 (2013).
Acknowledgements
J.O.A. was supported by a scholarship from CONACyT (scholar 213546) and P.K. by an ERC Starting Investigator Grant (Nanoscope, 337757).
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Arroyo, J., Kukura, P. Non-fluorescent schemes for single-molecule detection, imaging and spectroscopy. Nature Photon 10, 11–17 (2016). https://doi.org/10.1038/nphoton.2015.251
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DOI: https://doi.org/10.1038/nphoton.2015.251
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