Fluorescence Correlation Spectroscopy of Single Dye-Labeled Polymers in Organic Solvents Heiko Zettl, † Wolfgang Ha 1 fner, † Alexander Bo 1 ker, † Holger Schmalz, ‡ Michael Lanzendo 1 rfer, ‡ Axel H. E. Mu 1 ller, ‡ and Georg Krausch* ,† Physikalische Chemie II and Makromolekulare Chemie II, Universita ¨ t Bayreuth, 95440 Bayreuth, Germany Received December 17, 2003 ABSTRACT: We discuss the use of fluorescence correlation spectroscopy (FCS) to study the diffusion of single dye-labeled polymer chains in organic solvents. Monodisperse batches of polystyrenes labeled with a single Rhodamine B molecule have been synthesized via anionic polymerization of styrene and ethylene oxide end-capping followed by a polymer analogous coupling reaction. MALDI-ToF mass spectrometry is used to characterize the resulting material. A commercial FCS system has been modified to permit FCS measurements in volatile organic solvents. FCS was used to determine the molecular weight dependence of the diffusion coefficient of 10 nM solutions of end-labeled polystyrenes in toluene. The data are utilized to establish a calibration procedure for FCS measurements in organic solvents. Introduction Experimental techniques based on the detection of single molecule fluorescence have gained increasing interest throughout the past decade. They offer the unique opportunity to study the structural and dynami- cal properties of single molecular entities and circum- vent the loss of information inherent of ensemble averaging typical for most of the classical experimental techniques, requiring the detection of a large number of molecules. Prominent examples of this class of techniques are single molecule microscopy, single mol- ecule spectroscopy, and fluorescence correlation spec- troscopy (FCS). Despite their scientific beauty and their high potential, however, the use of single molecule fluorescence techniques in polymer science has almost entirely been limited to the area of biological macro- molecules and aqueous media. 1-3 This is even more astonishing, as a variety of potential applications in polymer materials science can be envisaged: the ultra- high sensitivity of FCS experiments could be utilized to study molecular self-assembly in solution in systems exhibiting extremely low critical aggregation concentra- tions (e.g., block copolymers, nanoparticles, etc.), and single molecule microscopy could be used to study the elementary processes of polymer crystallization or the movement of single chains in confined environment (polymer networks, ordered polymeric mesophases, thin films, etc.). Finally, single molecule spectroscopy could be used to follow the segmental dynamics of polymer chains via the time-resolved detection of changes in their immediate environment. Very recently, first ap- plications of the type mentioned above have been reported, including the self-aggregation of complex block copolymer aggregates 4-6 via FCS, the visualization of single dye-labeled polymer chains by single molecule microscopy 7 and FCS in water, 8,9 and the combination of FCS and surface forces measurements for the study of dye molecules confined to ultrathin films. 10 For single molecule fluorescence experiments to be used on synthetic polymers in organic solvents, a variety of problems have to be solved. At first, well-defined test polymer chains containing a single entity of a suitable dye have to be synthesized. Quite importantly, the resulting dye-labeled polymer must not contain any measurable amount of free dye molecules, so careful separation steps are crucial. Moreover, the issue of photostability of the dye has to be addressed both in view of the organic environment and in view of the fact that the time scales of molecular motion in concentrated polymer solutions and melts are considerably larger than in most aqueous systems studied so far. In addi- tion, most organic solvents have a considerably higher vapor pressure than water. Therefore, solvent-tight sample chambers need to be developed. The beam path of the optical setup has to be adapted to the index of refraction of the solvent, which proves difficult for most commercial experimental setups, which happen to be optimized for an aqueous environment. The above considerations hold for any kind of optical single molecule experiment in organic environment. For fluorescence correlation spectroscopy (FCS) in particu- lar, an additional problem occurs. Changes in the optical setup typically come along with changes in the dimen- sions of the detection volume. Therefore, suitable cali- bration procedures have to be devised. FCS calibration in aqueous environment typically relies on the straight- forward measurement of the characteristic diffusion time of a dye molecule with known diffusion coefficient. In organic media, however, no such simple means is available. Most dye molecules tend to aggregate in organic solvents, and in contrast to aqueous systems the diffusion coefficients are barely known. This problem can be circumvented if well-soluble objects of known hydrodynamic radius are labeled with a single dye molecule. 11 Alternatively, luminescent semiconductor nanoparticles of narrow size distribution and known hydrodynamic radius could be used. 12 Finally, single dye molecules may be linked to polymer chains of known molecular weight and narrow molecular weight distri- bution. In these cases, either the diffusion constant will be known or it can easily be determined from classical experiments like dynamic light scattering. In the present article, we describe first experiments along these lines. We have synthesized monodisperse † Physikalische Chemie II. ‡ Makromolekulare Chemie II. * Corresponding author. 1917 Macromolecules 2004, 37, 1917-1920 10.1021/ma035929t CCC: $27.50 © 2004 American Chemical Society Published on Web 02/04/2004