Nanoparticle-Induced Fluorescence Lifetime Modification as Nanoscopic Ruler: Demonstration at the Single Molecule Level J. Seelig, ² K. Leslie, ‡ A. Renn, ² S. K 1 uhn, ² V. Jacobsen, ² M. van de Corput, ‡ C. Wyman, § and V. Sandoghdar* ,² Laboratory of Physical Chemistry, ETH Zu ¨rich, 8093 Zu ¨rich, Switzerland, Department of Cell Biology and Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands, and Department of Radiation Oncology, Erasmus Medical Centre, Rotterdam, The Netherlands Received November 25, 2006; Revised Manuscript Received January 30, 2007 ABSTRACT We combine interferometric detection of single gold nanoparticles, single molecule microscopy, and fluorescence lifetime measurement to study the modification of the fluorescence decay rate of an emitter close to a nanoparticle. In our experiment, gold particles with a diameter of 15 nm were attached to single dye molecules via double-stranded DNA of different lengths. Nanoparticle-induced lifetime modification (NPILM) has promise in serving as a nanoscopic ruler for the distance range well beyond 10 nm, which is the upper limit of fluorescence resonant energy transfer (FRET). Furthermore, the simultaneous detection of single nanoparticles and fluorescent molecules presented in this work provides new opportunities for single molecule biophysical studies. Optical methods provide versatile means for measuring distances, ranging from astronomical to angstrom dimensions. At the small scale, the wave nature of light and the principle of diffraction hamper resolving two point objects that are separated by d < λ/2NA, where λ is the wavelength of light and NA denotes the numerical aperture of the optics used. However, it has been shown that measurements in the near field can get around this diffraction limit because one has access to high spatial frequency components of the optical field. 1-3 In standard near-field microscopy, one scans a sharp probe to interrogate the near fields of a sample and is therefore limited to surface studies. An alternative for accessing near-field interactions is to integrate the “probe” into the sample so that a far-field reading of some property of the probe provides information about its near-field interaction with the object of interest. This is indeed the underlying principle of fluorescence resonant energy transfer (FRET), 4 where two fluorophores undergo dipole-dipole coupling so that the energy of the one (donor) is transferred to the other (acceptor). One can therefore learn about the separation of the donor and the acceptor by examining the amount of emission from the latter. Because the efficiency of this process scales as 1/(1 + (r/r 0 ) 6 ), it is significant over a very short distance range of r 0 ∼ 4-8 nm for typical fluorophores. 5 Another configuration of a built-in “nanoscopic ruler” exploits the near-field interaction of two metallic nano- particles. 6,7 Although in general the interaction of the two particles is quite complex when considering all illumination polarizations, distance ranges, and particle sizes, the situation for very small particles can be understood as follows. Light induces oscillating dipole moments in each gold particle, and their instantaneous (1/r) 3 coupling results in a repulsive or attractive interaction, modifying the plasmon resonance of the system. The softer dependence of the interaction strength on the particle separation r results in a much longer interaction range compared to FRET. Very recently, a third possibility has been proposed in which the strength of fluorescence quenching and the accompanying change in the fluorescence lifetime act as measures for the separation between a fluorophore and metal clusters of diameter 1.5 nm. 8,9 In this case, sensitivity in displacements up to about 10-15 nm was demonstrated in ensemble measurements. Here we propose a generalized form of nanoparticle-induced lifetime modification (NPILM) and demonstrate its feasibility at the single molecule and single particle level. Our calcula- * Corresponding author. E-mail: vahid.sandoghdar@ethz.ch. ² Laboratory of Physical Chemistry, ETH Zu ¨rich. ‡ Department of Cell Biology and Genetics, Erasmus Medical Centre. § Departments of Cell Biology and Genetics and of Radiation Oncology, Erasmus Medical Centre. NANO LETTERS 2007 Vol. 7, No. 3 685-689 10.1021/nl0627590 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/23/2007