Two-Photon Fluorescence Microscopy of Single Semiconductor Quantum Rods: Direct Observation of Highly Polarized Nonlinear Absorption Dipole Eli Rothenberg, Yuval Ebenstein, Miri Kazes, and Uri Banin* Department of Physical Chemistry, The Farkas Center for Light Induced Processes and the Center for Nanoscience and Nanotechnology, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel ReceiVed: December 22, 2003 Two-photon polarization fluorescence microscopy is used to study the nature of the emission and nonlinear absorption dipole of single CdSe/ZnS quantum rods. Rods showed strongly polarized nonlinear excitation with sharp angular dependence, following a cos 4 (φ) functional form, in agreement with the predicted two- photon absorption process. The two-photon absorption is parallel to the emission polarization and allows high orientation selectivity in excitation to be achieved. This further demonstrates the role of single molecule measurements in unraveling basic principles of light-matter interactions otherwise masked by ensemble averaging. Two-photon absorption is a powerful tool for spectroscopy that allows excited electronic states in molecules, solids and nanostructures to be explored that cannot be excited in the single photon transition due to the different selection rules. 1-4 Two- photon microscopy exploits the nonlinear excitation process in achieving focal plane selectivity with improved signal-to-noise ratio, and is widely applicable to biological environments. 5 Previous studies of two-photon excited fluorescence of en- sembles in solutions showed superior sensitivity to molecular anisotropy, relative to linear absorption, as a result of the directionality induced by the two-photon interaction. 6 The fluorescence intensity in this case is proportional to |EE:T| 2 (eq 1) yielding enhanced angular selectivity compared to the linear absorption case proportional to |μ b‚E B| 2 (eq 2), where E B is the polarization of the exciting light, T is the two-photon absorptivity tensor, and μ b is the transition dipole moment. 7,8 Single molecule fluorescence provides powerful means for unraveling phenomena that are otherwise masked by ensemble averaging. 9-12 Of particular elegance is the direct observation of the linearly polarized transition dipole moment verifying eq 2 and allowing directional selectivity in molecular and nano- structure excitation. 13-16 Two-photon excitation of single mol- ecules was also reported, enabling statistical analysis of their orientation. 8 However, direct observation of the expected angular dependence derived from eq 1, for a single chromophore, was not reported yet. This may be due to the small two-photon cross- section and rapid photobleaching typical for dye molecules that were studied previously. 5 Here we report a study of the polarization nature of single semiconductor quantum rods (QRs) under two-photon excitation allowing to directly probe the angular dependence of single chromophores. QRs exhibit electronic and optical properties different than quantum dots (QDs). 17,18 Of particular importance to the present work, unlike the spherical dots, 19 QRs have linearly polarized absorption and emission, as demonstrated recently by fluores- cence measurements on single rods 20,21 consistent with theoreti- cal calculations. 22 The linear polarization also leads to the observation of polarized lasing from rods. 23 QRs are suitable for this study since they should have large two-photon cross sections as was reported for QDs, 5 and photobleaching is significantly reduced in such systems. 24,25 We find that the two- photon absorption of single QRs is linearly polarized with an angular dependence verifying eq 1 and distinctly different from the linear absorption case. To the best of our knowledge, this provides a first direct experimental confirmation of the unique nature of the two-photon transition dipole on the single molecule level. Samples studied here are CdSe/ZnS core/shell quantum rods of three sizes prepared using the well-developed methods of colloidal nanocrystal synthesis utilizing high-temperature py- rolysis of organometallic precursors in coordinating solvents as detailed elsewhere. 17,26,27 The thin ZnS shell was grown on the core rods to enhance the fluorescence quantum yield (QY) and stability, 23,28,29 while the outer surface is overcoated by organic ligands. The three samples were of dimensions 4 × 11 nm, 4 × 24 nm, and 4 × 60 nm (diameter × length) with QY of 30%, 24%, and 7% respectively. Transmission electron microscopy (TEM), as shown in the left inset of Figure 1 for the 4 × 24 nm sample, was used for size determination as well as to confirm the homogeneous rod shape. For single rod fluorescence measurements, an extremely dilute toluene solution of QRs was spin cast onto a pre-cleaned quartz substrate. Single particle fluorescence was measured on a locally built micro-PL setup. Fluorescence images were collected in an epi- illumination configuration, with a high numerical aperture long working distance objective (100× magnification, NA ) 0.70) under wide-field illumination. For the two-photon excitation source, we used a CW-mode locked Ti-sapphire laser (Coherent Mira) at 820 nm, with 120 fs pulse width (fwhm) and a 76 MHz pulse repetition rate. The images were focused onto the entrance of the monochromator, while the excitation laser was removed by a short-pass filter. Images and spectra were collected by a thermoelectrically cooled CCD (Lavision Imager QE) using a mirror and an appropriate grating, respectively. Both parallel and perpendicular polarization components were imaged simul- taneously using a polarization-displacing cube. Polarization of emission and excitation (laser) were rotated by the use of half wave plates at the appropriate wavelengths. * To whom correspondence should be addressed. E-mail: banin@ chem.ch.huji.ac.il. 2797 J. Phys. Chem. B 2004, 108, 2797-2800 10.1021/jp037978d CCC: $27.50 © 2004 American Chemical Society Published on Web 02/07/2004