NANO LETTERS
Wide-Field Optical Imaging of Surface Nanostructures
2006 Vol. 6, No. 7 1384-1388
Dominique Ausserre´* and Marie-Pierre Valignat CNRS UniVersite´ du Maine, UMR 6087, 72085 Le Mans Cedex 9 France, and INSERM UMR 600, CNRS UMR 6212, Aix-Marseille UniVersite´ , 13288 Marseille, France Received February 16, 2006; Revised Manuscript Received April 27, 2006
ABSTRACT We present a new technique that increases the sensitivity of incoherent light optical microscopy to a point where it becomes possible to directly visualize ultrathin films (∼nanometers) and isolated nano-objects. The technique is based on the use of nonreflecting substrate surfaces for cross-polarized reflected light microscopy. These surfaces generate a contrast enhancement of about 2 orders of magnitude, extending the application field of wide-field optical microscopy toward the nanoworld. The efficiency of the method is proven experimentally on well-characterized samples. Wide-field imaging of a nonlabeled λ-DNA molecule is also presented.
Reflected light optical microscopy is a well-established, lowcost, and widely used technique for probing surface structures and thin-film properties. In particular, working with reflected diffential interference contrast (DIC)1-3 allows one to probe surface topography and reveal the presence of nonlabeled tiny objects down to diameters of 40 nm. A number of methods have been proposed to further increase the image contrast in reflection microscopy.4-6 The basic idea is to get rid of the background image whose brightness depends on the reflectivity of the surface. This is often achieved by inducing a modulation of the polarization and using video image processing7 on the microscope image. Image contrast, however, cannot be generated where none, or little, exists. In bright-field microscopy, the raw contrast in the direct microscope image depends on the optical properties of the surface. The best contrast is achieved when the bare substrate surface does not reflect light, but with an added thin film or other object of observation it does. Blodgett and Langmuir8,9 in the mid 1930s were the first to study the change of reflectivity due to the presence of thin organic films at a substrate surface. They showed that thin organic films on polished chromium surfaces can be observed if they are applied not directly to the naked surface but to a barium stearate film first deposited on the chromium surface. The barium stearate film changes the reflectance of the substrate and produces interference colors. Sandstro¨m et al.10 have discussed such contrast slides in general. These studies made use of the properties of an antireflection (AR) layer. In the case of white light, an interference color is produced by the surface of the layered substrate. The presence of an additional film is then detected easily by the change in that color. * Corresponding author. E-mail:
[email protected]. 10.1021/nl060353h CCC: $33.50 Published on Web 06/21/2006
© 2006 American Chemical Society
The contrast, C, between a film and a surface is usually defined as the relative difference between the film intensity IF and the bare surface intensity IS, C ) (IF - IS)/(IF + IS). It is optimal (C ) 1 or C ) -1) if the intensity of either the substrate or the film goes to zero. Therefore, we shall first develop the expression for the intensity in the image of a planar reflecting surface. The illumination is spatially incoherent and for the sake of simplicity we assume a single incidence angle θ0 and a quasi-monochromatic wavelength, λ. Light beams impinging the substrate generate the surface of a cone (Figure 1). Each azimuth, φ, with respect to an arbitrary axis, x, on the cone defines a particular plane of incidence. The surface reflection is characterized by the two Fresnel coefficients, rp and rs, respectively parallel and perpendicular to that plane (sign conventions are the ones adopted by Azzam and Bashara12). We consider an isotropic surface, so that rp and rs are independent of the azimuth. Because the source is incoherent, two different beams reflected by the sample cannot interfere and the light intensity in the image is obtained by adding intensities of the backcoming beams. We define the reference intensity, I0, as the one obtained with the same incident beam and for a perfectly reflecting surface (rs ) -rp ) 1) and no polarizer. The normalized intensity R(x,y) in the image is the ratio R(x,y) ) I(x,y)/I0, where I(x,y) denotes the local intensity. When the incident beam is not polarized, every azimuth contribution is the same and the normalized light intensity RNP(x,y) in the image is the average of the square modules of the two complex Fresnel coefficients of the corresponding point on the surface RNP ) 1/2(|rp|2 + |rs|2). The antireflecting surface is defined by RNP ) 0, which requires both rp ) 0 and rs ) 0.These are the classical AR conditions. They can only be met with normal incidence. The single layer solution
Figure 1. Geometry and sign conventions. Episcopic illumination; enlargement: collimated ring beam of light focused by the objective lens to a spot on the sample. The directions of the x and y surface axes are chosen parallel, respectively, to the polarizer and to the analyzer. The p and s polarization components are displayed for a plane of incidence of azimuth, φ, relative to the direction of the polarizer. φ is the angle between the analyzer and the polarizer, here equal to π/2.
(AR layer) is defined by11 n1e1 ) λ/4(1 + 2m), where e1 is the layer thickness, m is an integer, and n1 ) xn0n2, where n0, n1, and n2 are, respectively, the refractive index of the ambient medium, the layer, and the substrate. To have a maximum contrast (C ) 1), the AR conditions must be fulfilled for the layered substrate (IS ) 0) and must be lost in the presence of the sample film (IF * 0). When the incident beam is linearly polarized along x, the radial symmetry of the illumination is broken and each azimuth, φ, brings a different contribution to the image intensity. As a consequence, most often the linear polarization of the reflected beam is lost. This depolarization upon reflection is the one exploited in ellipsometric measurements.12 Although the beam is incoherent, the two amplitude components, rp and rs, have a well-defined phase relationship and may interfere. The interference is concretized by interposing an analyzer in the reflected beam making an angle π/2 with the first polarizing plate. After averaging over all azimuths, the resulting intensity, R, is given by R)
1 |r + rs|2 16 p
(2)
This relationship is much less restrictive than the two classical AR conditions rp ) 0 and rs ) 0. Because of this Nano Lett., Vol. 6, No. 7, 2006
λ e1xn21 - n20 sin 2 θ0 ) (1 + 2m) 4 n21 ) n20
n22 + xn22 cos2 θ0(n22 - n20 sin 2 θ0) n22 + n20 cos2 θ0
(3a)
(3b)
These solutions are obtained for nonnormal incidence, which is an important feature for imaging. When considering small aperture, eqs 3a and b may be expanded at first order in θ0, reducing to
(1)
This signal is the one that was used in ellipsocontrast microscopy13 for sensitive imaging of thin films (∼10 nm) on regular silicon substrates. Between crossed polarizers, the intensity is always zero in normal incidence (rp + rs ) 0 with or without a film) so the AR supports do not work anymore for contrast enhancement. It is therefore natural to look for surfaces being antireflecting when working at nonzero incidence with polarized light (despite being nonAR with nonpolarized light). Setting the intensity to zero in eq 1 gives a single relationship between the two complex Fresnel coefficients r p + rs ) 0
additional degree of freedom, eq 2 has many solutions and defines a broad family of new surfaces (referred to as ARX-Pol) that can be used as working stages for imaging between crossed polarizer. Single layer solutions with nonabsorbing materials are obtained readily from the theory of light reflection. Similar to classical AR layers, each solution is given by two equations defining the layer thickness, e1, and the layer refractive index, n1
λ n1e1 ) (1 + 2m) 4 n21 )
2n20n22 n20 + n22
(4a)
(4b)
Let us emphasize that extinction conditions 3 or 4 are critical so that every film or object deposited on the surface restores the reflected light and appears bright on a dark background. As a consequence, the use of AR-X-Pol surfaces results in a striking amplification of the optical contrast for observation between crossed polarizers. Figure 2 illustrates the behavior of the theoretical contrast in a particular case where the AR-X-surface is made of a single layer standing with thickness e1 on a silicon surface (n2 ) 4.06-0.01i). The curves show the contrast obtained with a surface step 1385
Figure 2. Contrast of a 0.1-nm-thick film on a silicon solid bearing a single silica layer of thickness e1 as a function of e1; λ ) 570 nm. (A) Single incidence θ0 ) 30°, n1 ) 1.33. (B) Full cone microscope illumination with N.A. ) 0.5 and n1 ) 1.35 (bold curve) and with N.A. ) 0.7 and n1 ) 1.31 (thin curve).
of height ∆e1 ) 0.1 nm as a function of the layer thickness e1. The light wavelength is set to λ ) 570 nm. The contrast is almost perfect (C ) 1) for a single incidence illumination (here arbitrarily chosen equal to 30°) when the optical properties of the layer obey eq 4, that is, n1 ) 1.33 and e1 ) 115 nm. However, for visualization with an optical microscope, the relevant contrast should take into account all angles belonging to the incident light cone. Equation 4 cannot be satisfied for all incidence angles so that the extinction quality becomes poorer as the numerical aperture (N.A.) of the objective is raised. Nevertheless, an optimal value of the refractive index can be approached numerically starting from the value given by eq 3. It is assumed that sin θ0 d θ0 is the weight of each incident angle, θ0, in the image intensity. For instance, for N.A. ) 0.5, the calculated optimal properties of the layer are n1 ) 1.35 and e1 ) 113 nm. Then, the maximum contrast obtained is 3% and the submolecular step ∆e1 will be clearly visible in the microscope. The theoretical contrast is enhanced by a factor of 360 when compared to that of the same step observed on a conventional silicon substrate bearing a 2 nm native oxide layer (n1 )1.46), which is C ) 0.0083%. Notice that eqs 3 and 4 were established for dielectric media and that we have neglected the adsorption part k2 of the silicon refractive index when calculating n1. We checked numerically that taking k2 into account was not affecting the result. The sensitivity of the technique was illustrated quantitatively by imaging a stair-like sample (Silios, France) built on a silicon substrate covered with a 106-nm-thick silica layer. Although its refractive index is not optimal (1.46 instead of 1.35), the layer thickness is optimized and the substrate is a reasonable approximation of an AR-X-Pol surface. Indeed, from theory, the contrast value for the detection of a 0.1-nm-thick step is 85 times larger than the one obtained with a conventional silicon substrate. The stair 1386
is made of seven silica steps whose altitudes with respect to the oxide layer are, respectively, 2.0, 10.1, 18.3, 22.3, 30.7, 37.6, and 41.4 nm, as measured by ellipsometry. The silica layer and the steps where thermally grown at the same time on the silicon substrate. The steps were obtained by tuning locally the oxide growth rate using different time exposures of the silicon to appropriate ion beam prior to oxidation. Figure 3 shows two images of these steps as seen directly in the microscope (LEICA DMR HC, white light, Obj. PlanApo ×50, N.A ) 0.5). In Figure 3A, they were simply observed between cross polarizers. Without any image processing, each step was clearly visible and had a different color depending on its thickness. In Figure 3B, a DIC device was added to the microscope. Observation in the DIC mode allows one to clearly visualize the edges of the sample steps even for the thinnest one with 2 nm thickness. In addition, the brightness contrast between the different steps appears to be very important. Although a quantitative analysis of DIC contrast enhancement by AR-X-Pol surfaces is far beyond the scope of the present paper, we will now give a qualitative explanation of the observed effects. The DIC technique14 is designed to visualize thickness or index gradients and to detect objects smaller that the diffraction limit. The principle is that each incident beam is linearly polarized using a first polarizer B P and then split up into two beams with the same amplitude and orthogonal polarization directions making an angle (π/4 with the first polarizer. This is achieved with the help of the DIC device, a biprism made of birefringent materials and located in the rear focal plane of the objective. When emerging from the DIC device, the two beams have slightly different propagating directions. Consequently, after passing the objective lens, the two beams are laterally shifted with respect to each other by a small amount ∆r b along the sample. The shift direction is parallel to the polarization of one of the two beams, hence named the parallel component. When reflecting at the surface, the two beams experience different optical paths due to sample topography. After reflection, they are gathered by the objective lens and focused back on the DIC device where the lateral shift is eliminated. They finally interfere when passing through the analyzer B A, oriented orthogonal to the first polarizer. If the two components were unaltered by the reflection on the surface, then the initial polarization imposed by B P would reconstruct exactly the same and no signal would emerge from B A. However, the optical path difference between the two beams results in a phase difference that impedes this unaltered reconstruction. The reconstructed polarization is no more linear along B P. It is elliptic, and this results in a nonzero signal along B A. To optimize edge and nanoobject detection, it is usefull to work over a dark background. Background extinction is obtained by suppressing the ellipicity of the recombined polarization in flat regions of the sample with the help of a phase-compensating component, which is part of the DIC device. In practice, this so-called “bias retardation” tuning is ensured by moving laterally the DIC device along the shear direction. Notice that “parallel” here is relative to the DIC device geometry and has no relation with the “parallel”, or p, Fresnel coefficient. To summarize, the Nano Lett., Vol. 6, No. 7, 2006
Figure 3. Stairs made of steps with length 50 µm, width 10 µm, and respective altitude 2.0, 10.1, 18.3, 22.3, 30.7, 37.6, and 41.4 nm, from the bottom to the top. (A) Between cross polarizer; (B) with additional reflection DIC contrast. The two fist steps would not even be detected on regular substrates.
phase shift due to the instrumental optical path difference between the two beams is compensated while the phase shift associated with the local sample topography is revealed on a dark background by the analyzer as a “depolarization” intensity. The important point to consider is that a DIC image reveals depolarization components. The classical description given above assumes implicitly that the two beams generated by the DIC device remain linearly polarized when reflecting on the surface. This is not correct, however, as underlined all along in the present paper. In general, each of the two beams generated by the DIC device for a given incidence and azimuth is itself elliptically polarized after reflection on the surface due to amplitude and phase differences in the rp and rs Fresnel coefficients. This results in strong additional contributions along the analyzer. These contributions are imposed by the nature the sample surface, they are similar for the two beams (after azimuth averaging), and they cannot be compensated with the help of any existing tuning component in the microscope. Hence, in general they act as parasitic light and are responsible for contrast degradation in DIC observations. On AR-X-Pol surfaces on the contrary, they are responsible for contrast enhancement. Indeed from their properties, AR-XPol surfaces can also be defined as nondepolarizing surfaces, ensuring perfect and also critical background extinction. Because of better extinction, DIC becomes more efficient in edge and nano-object imaging. Contrast is also enhanced for thin-film and nano-object imaging because depolarization with surface reflection is restored as soon as a very small amount of material is present on the surface, acting as an optical switch. This is clearly illustrated in Figure 3b where the edges and the roughness of steps with thickness in the nanometer range are easily visible without any image processing, and where flat steps in the nanometer range can be easily differentiated from each other from their brightness. The sensitivity of the observation in the DIC mode was also tested on nanometric gold deposits obtained by electronbeam lithography.15 These structures cannot be visualized Nano Lett., Vol. 6, No. 7, 2006
Figure 4. Wide-field DIC image of gold nanometric deposits. (A) Dots 50 nm wide and 50 nm high. (Β) Bands with length 3 µm, width 50 nm, and thickness 5 nm. Bottom images are enlarged details of the corresponding main image.
when laying on a conventional silicon surface either in polarization microscopy or in DIC microscopy. Figure 4 displays the image of these nanometric deposits laying on an AR-X-Pol surface (a silicon wafer bearing a 106-nmthick layer of silica) as seen in DIC mode. The shape, the lateral size, and the height of the objects were probed by combining low vacuum scanning electron microscopy and tapping atomic force microscopy. Figure 4A shows dots 50 nm wide and 50 nm high. The apparent diameter of these objects is just the lateral resolution of the microscope, about 10 times larger than their true diameter. The bottom images in Figure 4A show enlargements of deposition defects, which were detected easily. The shape of 3 µm long, 50 nm wide objects is also visualized clearly in Figure 4B. The thickness of these flat objects is only 5 nm. Therefore, the section of the objects is about (16 nm)2. The small period of the pattern is 700 nm, which implies that the lateral resolution obtained is better than 350 nm. In the two previous examples, where dense surface patterns were imaged, we were actually still far from the detection 1387
Figure 5. Single phage-λ double strand DNA molecule with length 16.4 µm and diameter 2 nm. (A) Wide-field optical (DIC) image before AFM scanning; (Β) AFM image; (C) wide-field optical (DIC) image after AFM scanning.
limits of the technique. These limits are better illustrated by the last example where the sample is a stretched isolated phage λ-DNA molecule deposited on a HMDS-modified Si/ SiO2 contrast-enhancing substrate according to Bensimon and al.16 combing method. Until now, DIC detection was limited to objects with a minimum diameter of approximately 40 nm, at least in the case of gold nanoparticles.17 Transmitted light detection of 25 nm filaments has also been reported when combining DIC with video-enhanced microscopy.18 For comparison, we demonstrate in Figure 5 that the new technique allows wide-field detection of an isolated organic nanowire with 2 nm diameter. Indeed, Figure 5 displays three images of the same nonlabeled DNA molecule: Figure 5B is an AFM image of the molecule obtained in the tapping mode, and Figure 5A and C are optical reflected DIC images of the same molecule before (Figure 5A) and after (Figure 5B) AFM scanning. From AFM, the molecule height was found to be 1.7 nm, which is in good agreement with the expected diameter value of 2 nm. Notice that Figure 3b also reveals the nanometer-scale damages induced by the AFM tip to the HMDS layer. In conclusion, we have presented a new family of antireflective surfaces designed for cross-polarized illumination. To keep the surface chemistry of the support close to the one of a microscope slide or a conventional silicon wafer, we have first chosen to approximate the AR-X-Pol surface by a silicon wafer bearing a λ/4 single layer of silica. Although the refractive index is not yet optimal, the contrast is already enhanced by 2 orders of magnitude. As shown by the theory, ring lightening and further optimization of the support will permit better enhancement of the contrast. A great advantage of those surfaces is that they can be associated to any technique working with cross-polarized light. A striking example, but not limited to, is when they are associated to DIC microscopy observations. In this mode, using the same surface samples as above, we have actually been able to detect particles as small as 10 nm in diameter, cylindrical objects down to 2-5 nm in diameter, and image
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films with subnanometer resolution. Molecular film imaging and nonlabeled single nano-object detection becomes possible with a technique combining the conviviality of an optical microscope with molecular sensitivity, opening the route to new analysis and diagnosis tools. Acknowledgment. We thank Pascal Royer for providing us with the electron beam deposits and pattern characterization and David Bensimon for training us on DNA combing methods. References (1) Lessor, D. L.; Hartman, J. S.; Gordon, R. L. J. Opt. Soc. Am. 1979, 69, 357-366. (2) Cogswell, C. J.; Sheppard, C. J. R. J. Microsc. 1992, 165, 81-101. (3) Preza, C.; Snyder, D. L. J. Opt. Soc. Am. A 1999, 16, 2185-2199. (4) Ooki, H.; Iwasaki, Y.; Iwasaki, J. Appl. Opt. 1996, 35, 2230-2234. (5) Holzwarth, G.; Webb, S. C.; Kubinski, D. J.; Allen, N. S. J. Microsc. 1997, 188, 249-254. (6) Holzwarth, G. M.; Hill, D. B.; McLaughlin, E. B. Appl. Opt. 2000, 39, 6288-6294. (7) Oldenbourg, R.; Mei, G. J. Microsc. 1995, 180, 140-147. (8) Langmuir, I.; Schaefer, V.; Wrinch, D. M. Science 1937, 85, 7680. (9) ) Blodgett, K. B.; Langmuir, I. Phys. ReV. 1937, 51, 964-982. (10) ) Sandstro¨m, T.; Stenberg, M.; Nygren, H. Appl. Opt. 1985, 24, 472479. (11) Born, M.; Wolf, E. Principles of Optics, 6th ed.; Pergamon: New York, 1980; p 64. (12) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light; North-Holland, New York, 1977. (13) Ausserre´, D.; Picart, A.-M.; Le´ger, L. Phys. ReV. Lett. 1986, 57, 2671-2674. (14) Pluta, M. AdVanced Light Microscopy: Specialized Methods; Elsevier: Amsterdam, 1989. (15) Grand, J.; Kostcheev, S.; Bijeon, J.-L.; Lamy de la Chapelle, M.; Adam, P.-M.; Rumyantseva, A.; Le´rondel, G.; Royer, P. Synth. Met. 2003, 139, 621-624. (16) Bensimon, A.; Simon, A.; Chiffaudel, A.; Croquette, V.; Heslot, F.; Bensimon, D. Science 1994, 265, 2096-2098. (17) Boyer, D.; Tamarat, P.; Maali, A.; Lounis, B.; Orrit, M. Science 2002, 297, 1160-1163. (18) Gelles, J.; Shnapp, B.-J.; Sheetz, M.-P. Nature 1988, 331, 450-453.
NL060353H
Nano Lett., Vol. 6, No. 7, 2006
