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Physica E 17 (2003) 537–540

www.elsevier.com/locate/physe

Imaging single metal nanoparticles in scattering media byphotothermal interference contrast

David Boyera, Philippe Tamarata ;∗, Abdelhamid Maalia, Michel Orritb, Brahim Lounisa

aCentre de Physique Mol�eculaire Optique et Hertzienne, CNRS et Universit�e Bordeaux I, 351 Cours de la Lib�eration,33405 Talence, France

bHuygens Laboratory, Universiteit Leiden, Postbus 9504, 2300 RA Leiden, The Netherlands

Abstract

We have developed a photothermal method for far-/eld optical detection of nanometer-sized metal particles, combininghigh-frequency modulation and polarization interference contrast. We can image gold colloids down to 5 nm in diameter,with a signal-to-noise ratio higher than 10. This is a considerable improvement over commonly used optical methods based onresonance plasmon scattering which, for background reasons, are limited to particles of more than about 40 nm in diameter.By adding 300 nm latex spheres in the sample, we also show that in addition to its intrinsic sensitivity, our photothermalmethod is totally insensitive to non-absorbing scatterers.? 2002 Elsevier Science B.V. All rights reserved.

Keywords: Gold nanoparticles; Absorption; Heat di9usion; Di9erential interference contrast

1. Introduction

It is now possible to detect, image, and study sin-gle nano-objects by purely optical methods. This newmicroscopy completely removes ensemble averag-ing, so that the heterogeneity of populations as wellas the dynamical ;uctuations of individuals come tolight. In particular, ambient optical detection of singlemolecules is a fascinating opening in material scienceand molecular biology [1].Organic dyes can be chemically graphted to a

biomolecule under study, and are often used as ;uo-rescent labels. Because of the wavelength change it

∗ Corresponding author. Tel.: +33-556846213; fax: +33-5568-46970.

E-mail address: ph.tamarat@cpmoh.u-bordeaux.fr(P. Tamarat).

involves, ;uorescence is a low-background method.The main drawback, however, is photobleaching,i.e. the irreversible photochemical process leadingfrom the excited ;uorophore to a non-;uorescentproduct. Nanocrystals of II–VI semiconductors (suchas CdSe/ZnS) have recently been used as ;uores-cent markers [2,3]. Although they resist bleachinglonger than dyes, their luminescence intensity blinks,and they are diGcult to functionalize in a controlledmanner.The ideal label should be durable and generate an

intense optical signal, but at the same time be as smallas possible, not to perturb the observed molecule tooseverely. Metal particles are very appealing optical la-bels because they do not photobleach, and do not opti-cally saturate at reasonable exciting intensities. Singlelarge metal nanoparticles have already been imagedin optical microscopy by means of various methods

1386-9477/03/$ - see front matter ? 2002 Elsevier Science B.V. All rights reserved.doi:10.1016/S1386-9477(02)00862-7

538 D. Boyer et al. / Physica E 17 (2003) 537–540

based on Rayleigh scattering [4–6]. In strong back-ground media like cells or tissues, it is impossible todetect particles with diameters below 40 nm, becauseRayleigh scattering decreases like the sixth power ofdiameter.Since the optical absorption of a small metal particle

decreases like the third power of its diameter only, ab-sorption prevails over scattering for small enough par-ticles. The absorption cross-section of a gold particleof 5 nm diameter is about 3 nm2 at 514 nm [7], morethan two orders of magnitude larger than that of an or-ganic ;uorophore at room temperature. This strong ab-sorption gives rise to a change in temperature aroundthe particle under laser illumination. In this work, wedemonstrate how to optically detect this temperaturechange by a sensitive interference measurement akinto DIC. Photothermal detection was proposed earlierby Tokeshi et al. [8], who used a thermal lens e9ect todetect very low concentrations of absorbing moleculesin liquid solutions. Instead, we use the very sensitivepolarization interference method [9] which allows thedetection of slight phase changes induced by heating.

2. Experimental

The experiment is performed with two lasers, a(green) argon ion laser at 514 nm for heating, and a(red) helium–neon laser at 633 nm for probing. Thegreen beam intensity is modulated at frequencies be-tween 100 kHz and 10 MHz by an acousto-opticalmodulator. The linearly, horizontally polarized redlight is injected by transmission through a polarizingcube (see Fig. 1). A Wollaston prism, rotated by 45◦

from the incident polarization, splits the red beam intotwo orthogonally polarized beams, with a splitting an-gle of about 1 mrad. The two beams are focused inthe object plane of the microscope objective (×100,NA = 0:95, for air) as two di9raction-limited spots,1:2 �m apart. The green beam is superimposed on oneof the red beams by a dichroic beam-splitter. Just as inthe usual DIC method, the two re;ected red beams re-combine in the Wollaston prism. The phase di9erencebetween probe beams gives rise to a vertically polar-ized component, which is now re;ected by the polar-izing cube, and sent to a fast photodiode, through ared-pass /lter to eliminate green stray light. A lock-inampli/er detects the variations of the red intensity and

Fig. 1. Schematic diagram of the optical setup. The horizontallypolarized red probing beam is split into two beams by a Wollastonprism and sent to the microscope objective via a telecentric lenssystem (not shown). The re;ected beams are recombined in theWollaston prism and the vertically polarized beam is re;ected andsent to the detector. The green heating beam is modulated at highfrequency by an acousto-optical modulator.

thus the dephasing between the two red beams at themodulation frequency of the green beam with an in-tegration time of 10 ms. Microscopic images are ob-tained by scanning the sample with respect to the threespots.We prepared the samples by spin-coating a drop

of an aqueous solution of poly-vinyl-alcohol (PVA,1 wt%) doped with gold nanoparticles, on a micro-scope cover slip. The gold particles had diameters of20, 10 and 5 nm with half-maximum dispersions indiameters of 10%.

3. Results and discussion

The volume undergoing a signi/cant temperaturemodulation is determined by the damping of heatwaves at the modulation frequency. The frequencyis chosen such that this volume roughly overlaps thefocal spot. Its value is given by !S = 2�=CR2S, where� and C are, respectively, the heat conductivity andthe volume heat capacity of the medium, and RS isthe green spot radius. For a PVA matrix, the !S valueis around 1 MHz.The three-dimensional representation in Fig. 2a

is a photothermal image of a 20 nm diameter goldnanoparticle, with an average heating intensity of7 MW=cm2. We also imaged 5 nm diameter parti-cles with a remarkably large signal-to-noise ratio of

D. Boyer et al. / Physica E 17 (2003) 537–540 539

Fig. 2. (a) Photothermal image of a 20 nm diameter gold parti-cle, with an average heating power of 20 mW and a modulationfrequency of 800 kHz. This image was obtained in 4 s with anintegration time of 10 ms per pixel. (b) Linear dependence of thesignal on the heating power. (c) The signal as a function of themodulation frequency, with a /t by a numerical simulation basedon heat di9usion.

more than 10 [10], and even 2:5 nm diameter parti-cles, with a signal-to-noise ratio of about 2. We builta histogram of peak heights for about 200 imagedspots, and obtained a fairly narrow unimodal distribu-tion, con/rming that the spots arise from individualnanospheres.To prove that the imagingmechanism is indeed pho-

tothermal, we /rst studied the dependence of the signalwith the particle size and the heating laser power. Thesignal intensities for 5, 10 and 20 nm diameter spheresvary linearly with the volume of the particles. The sig-nal is perfectly linear in the heating power (Fig. 2b)with no sign of saturation in the range of intensitiesused in the present experiments, up to 20 MW=cm2.

We then investigated how the signal depends on themodulation frequency. We expect a decrease of thesignal with frequency, especially for !¿!S, wherethe heat di9usion characteristic length becomes shorterthan the spot size. We observe this behavior, in fullagreement with numerical simulations based on heatdi9usion (Fig. 2c).For future uses of photothermal imaging in sin-

gle particle tracking or for the localization of labelledbiomolecules, it is important to investigate how thephotothermal image is a9ected by the presence ofstrong scatterers in the sample. For this purpose, we

Fig. 3. DIC (a) and photothermal (b) images of a sample containing80 nm diameter gold spheres, 10 nm diameter gold spheres, and300 nm diameter latex spheres. For (b), the heating intensity is1:5 MW=cm2. The 80 nm gold spheres appear on both images,showing that they stem from the same sample area. In the DICimage the 10 nm particles are utterly invisible, while the 300 nmlatex spheres give very strong signals and the 80 nm gold spheresweaker ones. On the photothermal image, the 80 nm gold spheressaturate the detection, and 10 nm particles appear clearly, whereasthe strongly scattering latex spheres are completely absent.

added large latex spheres as well-calibrated scatter-ers to the spin-coating solution. Fig. 3 shows imagesof a sample containing three types of particles: la-tex spheres with 300 nm diameter, gold spheres with80 nm diameter, and gold nanoparticles with 10 nmdiameter. The left image (Fig. 3a) is an ordinary di9er-ential interference contrast (DIC) image of the sample,showing scattering and absorbing structures. The latexspheres, being very strong scatterers, give intense sig-nals with positive and negative contrast. The 80 nmabsorbing gold spheres appear with negative contraston the image, but their intensity is much weaker thanthat of the latex spheres. The right image (Fig. 3b)is the photothermal signal with a heating power of1:5 MW=cm2. The 80 nm diameter gold spheres givea very intense signal and saturate the lock-in ampli-/er. They appear at the same location as in the DICimage. New, weaker spots from the 10 nm nanoparti-cles appear with a signi/cant signal in the image.The absence of latex spheres in the photothermal

image shows that our method is totally insensitiveto non-absorbing scatterers, even when they arelarge objects with strong index contrasts with theirsurroundings. Only absorbing objects with high sat-uration intensities will appear in the photothermal

540 D. Boyer et al. / Physica E 17 (2003) 537–540

image under our present conditions. In biologicalsamples in particular, the absorption background fromauto-;uorescence or ;uorescent labelling would becompletely negligible.

4. Conclusion

We have demonstrated the advantages of photother-mal detection for absorbing nano-objects in scatteringmedia, namely the absence of background, the absenceof any saturation for illumination intensities up to sev-eral tens of MW=cm2, and the absence of photobleach-ing. To our knowledge, no other method, not evennear-/eld optics [11], provides a better signal-to-noiseratio. Our method can be implemented with DIC on astandard optical microscope, and be applied to physi-cal chemistry and material science, as well as labelledbiomolecules tracking in cells or tissues.Further improvement of the method will be the use

of a transmission geometry with a second Wollastonprism. The better collection of the probe beam willlead to a signi/cant increase of the signal-to-noiseratio.

Acknowledgements

This work was supported by CNRS (Individ-ual Nano-Object program), RNegion Aquitaine, andthe French Ministry for Education and Research(MENRT).

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