Apertureless Near-Field Scanning Optical Microscopy of Single

Interplay of Quenching and Enhancement Effects in Apertureless Near-Field Fluorescence Imaging of Single Nanoparticles. Eyal Yoskovitz , Ido Hadar , A...
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NANO LETTERS

Apertureless Near-Field Scanning Optical Microscopy of Single Molecules

2004 Vol. 4, No. 7 1329-1332

Vladimir V. Protasenko and Alan C. Gallagher* JILA, UniVersity of Colorado and National Institute of Standards and Technology, Boulder, Colorado 80309-0440 Received April 8, 2004; Revised Manuscript Received May 26, 2004

ABSTRACT Single molecules within poly(vinyl alcohol) film are studied in a confocal, vacuum apertureless near-field scanning optical microscope. Fluorescence images show asymmetry, attributed to interference between evanescent and probe fields, combined with the direction of onedimensional absorption dipole of the molecule. A model for the evanescent and probe excitation fields qualitatively reproduces many observed features of molecule fluorescence images. In the experiment we measured up to 7.3 times fluorescence enhancement and ∼30 nm fluorescence resolution.

To overcome diffraction limited optical resolution (∼200 nm for oil-immersion objectives), two different methods are widely used today: (a) a small aperture provides subdiffraction excitation in the vicinity of the aperture; this nearfield scanning optical microscopy (NSOM) normally uses the end of a tapered fiber as a excitation source,1-3 and (b) a sharp elongated protrusion, such as the probe of an atomic force microscope (AFM), generates a strong and local field near the tip when illuminated by an optical field with polarization along its axis.4-6 The strength and confinement of the tip near-field depend on the probe shape and material, as well as on the type of excitation (evanescent, plane wave, or focused).7,8 The combination of an atomic force microscope with an optical collection system focused on the probe tip yields apertureless near-field scanning optical microscopy (ANSOM). ANSOM has been used in studies of carbon nanotubes,9 ferroelectric materials,10,11 quantum dots (QDs),12,13 and solid-state lasers,14 with elastic scattering, Raman signal, or fluorescence being detected. Previously, we studied ANSOM fluorescence from CdSe/ ZnS QDs, because of their good optical stability against bleaching.12,13 The QD fluorescence images were normally symmetric with respect to the axis of the evanescent wave propagation, and they included far-field positive and negative interferences, near-field fluorescence enhancement, and fluorescence quenching. Images were consistent with a nearly isotropic two-dimensional (2D) or three-dimensional (3D) absorption dipole and the 2D nature of the emitting dipole,18 but exceptions occasionally occurred when one side of a “dirty” probe severely quenched the fluorescence. In contrast, a one-dimensional (1D) dipole is expected to be more appro* Corresponding author. JILA, University of Colorado, UCB 440, Boulder, CO 80309-0440. Business Phone: (303) 492-7841. E-mail: [email protected]. 10.1021/nl049474c CCC: $27.50 Published on Web 06/19/2004

© 2004 American Chemical Society

priate for molecular excitation and fluorescence as has been already shown in confocal microscopy18 and NSOM.19 A 1D dipole can also produce quite different ANSOM fluorescence images that are sensitive to the dipole direction. Here we present ANSOM studies of fluorescent molecules and a qualitative explanation of the observed fluorescence intensity patterns. Because many molecule fluorophores bleach rapidly, and this is usually attributed to oxygen reactions, we decrease oxygen exposure by (a) studying fluorescing impurity-molecules within a thin poly(vinyl alcohol) (PVA) polymer film deposited on a glass substrate, and (b) performing the ANSOM experiment at 10-4 Torr residual-gas pressure. Under these conditions ∼20% of the molecules emit ∼108 photons before bleaching, yielding >1 h of data acquisition time at ∼200 W/cm2 of excitation intensity. These impurity molecules conveniently provided a wide range of dipole orientations and reasonable stability against bleaching. The origin of impurities is not well established, although some comes from filtered deionized water used to dilute the poly(vinyl alcohol) before spin coating. Although the molecular species are not known, the following observations indicate that these are single-molecule fluorophores. (a) The ANSOM-scanned fluorophores, which were among the brightest found, on average have fluorescence intensity ∼75% of that measured from single terrylene molecules and ∼30% of single Rhodamine 6G, all at 488 nm excitation. We estimate the fluorescence cross section of the studied impurities to be 0.3-1.2 Å2, based on the excitation power, optical collection efficiency and measured intensity of fluorescence, as explained in ref 12. The observed range of fluorescence intensities (and cross sections) is attributed to the variations of molecule orientation with respect to the excitation polarization and the variations in species. (b) The fluorophores

Figure 1. (a) Core idea of the confocal ANSOM. Fluorescence images are obtained with the same probe on two molecules: (b) and ((c), and (d)). Images (b) and (c) are taken at p-polarized excitation; image (d) is measured under s-excitation. Arrow K Bev shows the propagation of the evanescent wave. Scan (b) is 2.5 × 2.5 µm2, while (c) and (d) are 1 × 1 µm2. 256 × 256 pixel images are smoothed by a 3 × 3 averaging matrix.

blink and bleach in single steps. (c) The average brightfluorophore spacing was ∼5 µm, so the ∼30 nm diameter probe-enhancement region is very unlikely to contain multiple fluorophores. Those at greater separation are optically resolved. (d) As shown next, the character of the measured probe-induced fluorescence qualitatively fits a model for probe interaction with a single 1D dipole. Observations (b) and (d) in particular demonstrate that these are single fluorophores, not small aggregates formed by many molecules. A detailed description of the optical and AFM parts of our ANSOM apparatus has been published elsewhere,12 and only a sketch of the probe, fluorophore, and optical fields is presented in Figure 1a. The off-axis illuminated, oilimmersion objective yields p- or s-polarized, 488 nm evanescent excitation above the glass cover slip under totalinternal-reflection conditions. The same objective lens collects from 500 to 700 nm molecule fluorescence, which is detected with an overall efficiency of ∼10%. The AFM operates in noncontact mode with probe tip 5-10 nm above the surface vibrating with 1 nm rms amplitude, and the cover slip with fluorophores are raster scanned while the probe is normally maintained at the center of excitation and collection foci. The silicon probes, manufactured for intermittentcontact mode, are etched in buffered HF for improved ANSOM properties.12 The optics collects fluorescence from molecules within ∼1 µm diameter collection focus, and with sparsely dispersed molecules the fluorescence is usually detected from a single molecule. The AFM z-feedback produces topographic images of the sample surface with ∼0.5 nm vertical resolution and 20-30 nm width; the probe tip radius is nominally ∼5-10 nm. 1330

To lower the fluorescence background from the cover slip, it was preflamed with an acetylene torch and yielded no fluorescence. PVA polymer film was deposited on it from a PVA-water solution using a spin-on procedure, where a small amount of the PVA solution rested on the glass cover slip for a few minutes before the spin. A variable-angle spectroscopic ellipsometer indicated 2-4 nm average film thickness. In analysis of the probe-fluorophore optical coupling, it is usually assumed that the probe is made from a homogeneousmaterialwithoutsurfaceimpuritiesorirregularities.7,8,15-17 Such probes produce fluorescent images from CdSe/ZnS QDs or vertically aligned molecules that are symmetric with respect to the excitation direction, and this is often observed. The nearly spherical QDs are expected to absorb and radiate almost independently of excitation field direction,18 and most ANSOM images are consistent with this. However, some ANSOM images obtained from QDs with silicon probes are asymmetric,12,13 suggesting that these probes caused asymmetric fluorescence quenching and/or field enhancement. To prevent this possible source of molecule fluorescence asymmetry, we collected images with the same probe on different molecules. One set of such data is shown in Figure 1, where a probe scans one molecule in part (b), and a second molecule in parts (c) and (d). Parts (b) and (c) are taken with p-polarized excitation, where the major component of the optical field B Eev is normal to the substrate surface (nearly parallel to the probe axis), and a smaller, 90° phase-shifted component is parallel to the substrate and to the excitation K Bev propagation direction (y, vertical slow scan axis).20 Figure 1d is measured with s-polarized excitation, with the field in the plane of the substrate and orthogonal to the propagation direction (xdirected, horizontal fast scan axis). The arrow K Bev shows the propagation direction of the evanescent excitation, which is common for all images presented. In Figure 1b, the probe is at the center of a small bright spot, where ∼6 times fluorescence enhancement (FE) occurs when the molecule passes below it. (FE is defined as a ratio of molecule fluorescence intensity with the probe above a molecule and to that with retracted probe.) The FE spot is at the focus of several bright (dark) parabolas, produced by positive (negative) interference between the optical field of the evanescent wave and that scattered by the probe (phase shift between excitations equals ∼2(n + 1)π for bright and ∼(2n + 1)π for dark parabolas, n ) 0, 1, 2 ...). This fluorescence image is similar to images obtained from QDs [ref 13, Figures 1 and 3], although the fringe contrast is particularly strong here due to the off-center probe position within the fluorescence-collection region. Because of the similarity to QD images, the left-right symmetry, and the large FE in Figure 1b, we believe that it is produced by a nearly vertical (z-direction) molecular dipole. This is also consistent with computer simulations presented below. In contrast to Figure 1b, the images in Figures 1c and 1d show a major left-right fluorescence asymmetry. Topology images (not shown here) reveal insignificant lateral drift between Figures 1c and 1d, so differences between the Nano Lett., Vol. 4, No. 7, 2004

Figure 2. Measured, (a) and (c), and calculated, (b) and (d), fluorescence images for differently oriented absorption dipoles b µabs(x′,y′,z′). The numbers in images (b) and (d) show the orientation of the dipole taken for simulations. All scans have 1 × 1 µm2 size, and the system of coordinates is left-handed (Z arrow points out of image plane). Experimental images (256 × 256 pixels) are smoothed by a 3 × 3 averaging matrix.

location of FE in Figure 1c and quenching in Figure 1d are caused by the different excitation polarization. In Figure 1c, FE = 3 is measured at the bright spot, where the fluorophore is under the probe. A dark area of negative excitation interference and perhaps probe quenching occurs on only one side of the molecule. In Figure 1d FE = 1.8 is observed at the spot at the center of the scan, and ∼35% fluorescence decrease (relative to without probe) occurred in the dark area to the right and above the FE location. This “large” FE at s-polarization, as well as the asymmetry, is quite different from typical behavior observed on QDs [ref 12, Figure 7c, and ref 13, Figure 5], again indicating that these features are due to the directional character of a 1D molecular dipole. Images from a second probe and two different molecules, using p-polarized excitation, are shown in Figures 2a and 2c. Again, the nearly symmetrical image in Figure 2a is attributed to a nearly vertical molecular dipole direction and a symmetric probe. The asymmetric image in Figure 2c is attributed to a tilted molecular dipole. For better understanding of the images, we have evaluated a simple model for comparison to the p-polarized excitation data. The fluorescence is proportional to the rate of excitation times the fluorescence quantum efficiency. Evaluating the optical coupling between fluorescing dipole and a probe versus probe-molecule configurations is nontrivial, so as a first approximation we assume constant QY of a molecule near a probe and calculate only the excitation rate versus molecule position. This is proportional to |(E Bev + B Epr)‚µ babs|2, where b µabs is the optical dipole of the molecule, B Eev is the evanescent optical field (assumed unperturbed by the probe), and B Epr is the probe optical field at the molecule. The probe Nano Lett., Vol. 4, No. 7, 2004

field is assumed cylindrically symmetric about the probe axis and is represented by the near-field of a vertically elongated Si ellipsoid ( ) 15) [ref 7, eq 7], plus the full field of a vertical dipole 80 nm above the substrate [ref 21, Chapter 4], which yields the more distant field due to the upper portions of the probe. The dipole field on the surface is the sum of the dipole field and the field reflected by the surface, where the reflected field is based on the Fresnel formulas.22 The vertical dipole and internal ellipsoid field are assumed in phase with the incident field, since the phase of the evanescent wave does not depend on z and the dielectric constant of silicon is primarily real at 488 nm. The radius of curvature of the ellipse apex and the probe-surface gap was varied within the expected range (from 5 to 10 nm) to best reproduce the measured fluorescence patterns. For a vertically oriented absorption dipole, this model yields the image in Figure 2b, which approximately reproduces the measured (Figure 2a) shape and intensity of the FE (bright spot), plus the positive interference parabolas (1) separated from the molecule by negative interference parabolas (3). Area 2 in Figure 2a is where the molecule is experiencing the positive interference between direct and forward scattered probe fields, where the phase shift is zero between excitations. The FE depends on the molecular quantum efficiency and probe quenching of fluorescence.13 The measured FE of ∼6 and 2.3 in Figures 1a and 2a are in the range obtained from the model (FE ) 3.5 in Figure 2b), which includes only the enhancement of excitation intensity, not fluorescence quenching. The difference in fringe separation between Figure 2a and Figure 2b is attributed primarily to two reasons: (a) using a single dipole, placed 80 nm above the surface, to produce the probe far-field in the model; the dipole phase delay cannot fully represent the phase of the actual probe field, where each part of the probe contributes with specific phase delay, and (b) to the piezo scanner calibration procedure, where the scanner was calibrated with large scans and piezo nonlinearity has not been taken into account. The calculated fluorescence image for a tilted dipole is shown in Figure 2d, where the tilt is adjusted to visually simulate Figure 2c. The model reproduces the general character of the FE bright spot, plus the areas labeled 1, 2, and 3 in Figure 2c. It similarly explains the Figure 1c data as due to a different molecular dipole direction. However, this simple model also generates features that do not occur in the experiment, such as a narrow dark area between the FE location and area 2 with a tiny bright “bridge” between them, not seen on measured data (Figure 2c). In all images the region near the FE spot is more broadened in the measured scans, as expected due to the difference between the steepsided ellipse and the tapered and rough shape of the actual probe. In addition to a more accurate model for the probe field, orientation-dependent fluorescence quenching may be needed. ANSOM measurements of fluorescing molecules deposited with a thin PVA film reveal a strong dependence on the orientation of the molecular-absorption dipole. This clearly indicates the 1D character of a molecular dipole, in contrast 1331

to a quantum dot, as well as an ability to measure the dipole direction using ANSOM. A simple model for 1D dipole excitation by the probe plus evanescent fields explains many, but not all, features in the ANSOM data. Up to 7.3 times fluorescence enhancement at p-polarized excitation and ∼73% quenching at s-excitation have been measured. The best detected fluorescence resolution is ∼30 nm for enhancement and ∼35 nm for quenching. As a final note, we provide some statistical data on longlived molecules. (Many others bleached before full ANSOM scans were achieved.) Five of ten probes were either optically inactive or weakly interacting, or showed strong and broad quenching, similar to what we sometimes observed on QDs.12,13 Thus, the data obtained with these probes on 18 molecules are not reported here. The other five probes yielded useful ANSOM data scanning another 15 molecules, and a representative set of this is shown in the paper. Among these 15 molecules we detected seven fairly symmetrical ANSOM fluorescence images, and eight clearly asymmetrical. These also yielded fluorescence enhancements of 1.8-7.3, with 30-70 nm full-half-width optical resolution, and the quenching varied from 21% to 73%. As we noted previously, all probe-induced changes in molecule fluorescence are calculated with respect to fluorescence intensity with a retracted probe and an unperturbed molecule. Acknowledgment. The authors thank Prof. W. E. Moerner for providing terrylene. This work is supported by the Quantum Physics Division of the National Institute of Standards and Technology. References (1) Hecht, B.; Sick, B.; Wild, U. P.; Deckert, V.; Zenobi, R.; Martin, O. J. F.; Pohl, D. W. J. Chem. Phys. 2000, 112, 7761-7774.

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NL049474C

Nano Lett., Vol. 4, No. 7, 2004