500 OPTICS LETTERS / Vol. 33, No. 5 / March 1, 2008

Heterodyne holographic microscopy ofgold particles

Michael Atlan,1,* Michel Gross,1 Pierre Desbiolles,1 Émilie Absil,2 Gilles Tessier,2 andMaïté Coppey-Moisan3

1Laboratoire Kastler-Brossel de l’École Normale Supérieure, CNRS UMR 8552, Université Pierre et Marie Curie - Paris6, 24 rue Lhomond 75231 Paris Cedex 05, France

2École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS UPR 5, Université Pierre et MarieCurie - Paris 6, 10 rue Vauquelin 75231 Paris Cedex 05, France

3Département de Biologie Cellulaire, Institut Jacques Monod, CNRS UMR 7592, Université Paris 6 and 7,2 Place Jussieu, Tour 43, 75251 Paris Cedex 05, France

*Corresponding author: atlan@lkb.ens.fr

Received September 25, 2007; revised December 6, 2007; accepted December 27, 2007;posted February 4, 2008 (Doc. ID 87919); published February 28, 2008

We report experimental results on heterodyne holographic microscopy of subwavelength-size gold particles.The apparatus uses continuous green-laser illumination of the metal beads in a total internal reflection con-figuration for dark-field operation. Detection of the scattered light at the illumination wavelength on acharge-coupled-device array detector enables 3D localization of brownian particles in water. © 2008 OpticalSociety of America

OCIS codes: 180.6900, 090.1995, 170.0180.

Assessing microscopic processes by tracking opticallabels has a broad range of applications in biology. Inparticular, monitoring biological phenomena such ascellular-level dynamics is a subject of growing inter-est. Fluorescent molecules are widely used in thisaim, but the observation time of dyes is limited byphotobleaching. Quantum dots offer a much betterphotostability, but they have the inconvenience ofblinking, whereas noble metal nanoparticles have theadvantage of being perfectly photostable [1]. Origi-nally, light scattered by small metallic particles hasbeen detected in dark field [1] or total internal reflec-tion (TIR) [2] configurations. To improve the detec-tion sensitivity, interferometric approaches havebeen introduced [3–7]. Common-path interference,initially achieved with a Nomarski interferometer[8], translates phase variations into intensity varia-tions and enables the detection of phase perturbationprovoked by spatial [9] or photothermal [10] modula-tion. Scanning heterodyne detection of the photother-mal modulation [3] enables an unmatched combina-tion of sensitivity and selectivity suitable todiscriminate particles smaller than 5 nm from theirbackground. Photothermal imaging relies on a spa-tial scanning of the laser beams to track the indexmodulation in the neighborhood of the beads; thetemporal noise in the incident light leads to spatialnoise in the image acquired sequentially [7]. Wide-field detection schemes alleviate such issues, buttheir sensitivity in optical mixing configurationswould not match heterodyne detection on single-detector levels. We propose here a wide-field, shot-noise-limited, tunable CCD heterodyne-detectiontechnique able to achieve high-resolution, 3D micros-copy of gold particles with a laser source at ratescompatible with biological dynamics. The experimen-tal setup is shown in Fig. 1(a).

The main laser beam (�=532 nm, field EL, 50 mW,single axial mode, cw) is split with a polarizing beam

splitter (PBS) in local oscillator (LO) and illumina-

0146-9592/08/050500-3/$15.00 ©

tion arms (fields ELO and EI). A � /2 waveplate (HWP)and neutral densities (A1, A2) allow control of the op-tical power in each arm. Both beams are frequencyshifted around 80 MHz by acousto-optical modulators(AOM) driven at frequencies �AOM1

, �AOM2. The LO

beam passes through a beam expander (BE) to form aplane wave whose polarization angle is adjusted witha HWP to maximize the holographic modulationdepth. The object is illuminated in a dark-field con-figuration by using TIR. The scattered field E�ELOpasses through a microscope objective (MO, 60�magnification, NA=0.9, air). Off-axis optical mixing(� tilt) of E with ELO with a beam splitter (BS) resultsin a fringe pattern recorded with a CCD camera(PCO Pixelfly QE, 1392�1024 square pixels of6.7 �m, frame rate �S=12 Hz). Proper frequency de-tuning ��=�AOM2

−�AOM1and angular tilt � enable

accurate and sensitive phase shifting off-axis holog-raphy [11,12].

The first samples observed are thin layers of goldbeads (diameter d=50 to 200 nm) immobilized in apolyvinyl alcohol (PVA) matrix spread by spin coatingonto a glass slide. These slides are set onto a prismused to guide the illumination field EI and provokeTIR at the PVA–air interface. Microscope immersion

Fig. 1. (a) Experimental setup. (b) Evanescent wave illu-mination of static beads in a plane. (c) Total internal reflec-tion configuration for dark-field illumination of beads in a

tridimensional environment. Acronyms defined in the text.

2008 Optical Society of America

March 1, 2008 / Vol. 33, No. 5 / OPTICS LETTERS 501

oil between the prism and the slide enables refractiveindex matching. The evanescent wave locally frus-trated by the beads is turned into a propagative scat-tered field E collected by the MO. To make E and ELOin phase opposition from one frame to the next, two-phase detection ���=�S /2� is performed. Exposuretime is �E=50 ms, and holograms are obtained bymaking the difference of consecutive frames. Thecomplex field E�x ,y ,z�, in the beads plane z, is recon-structed by numerical Fresnel transform [11].

Figure 2 shows the bead images [(a),(b),(c)] and thecorresponding intensity profiles along x [(d),(e),(f)].The images are obtained by averaging ��E�x ,y ,z��2�over four sequences of two images. The bead diam-eters are 200 [(a),(d)], 100 [(b),(e)], and 50 nm [(c),(f)].Under 50 nm the coherent parasitic light scatteredby dust or surface roughness prevents the beads to bedistinguished. Figure 3 shows, for 200 nm beads, thex, y images [(a),(b),(c)] and the x profiles [(d),(e),(f)],at z=0, 17, and 22 �m relative reconstruction dis-tances. It also shows the x, z image (g) and the z pro-file (h), in the z=0–47 �m range. Because the beademitters are close to a plane dielectric interface [13],one observes an asymmetric signal in the z directionof �7 �m width at half-maximum with rings in the x,y plane. Similar rings have been observed withquantum-dot emitters [14,15]. A quantitative study ofthis effect [13,16] is out of the scope of this Letter.

To illustrate the sensitivity of our holographicsetup, we have displayed on Fig. 4 images [�a� , . . . , �e�and �k� , . . . , �o�] and profiles [�f� , . . . , �j� and�p� , . . . , �t�] of 200 nm beads at different illuminationintensities. The illumination beam ��EI�2� is attenu-ated by a factor 100 [(a),(f),(k), and (p)], 101 [(b),(g),(l),and (q)]…, 104 [(e),(j),(o), and (t)]. The LO beam is ei-ther off [direct imaging; �a� , . . . , �j�], or on [holo-graphic regime, �k� , . . . , �t�]. The beads are imagedwith much better sensitivity with the LO beam, sincethe bead signal remains visible when the illumina-tion power is reduced by over 4 orders of magnitude.Moreover, the signal is proportional to illumination[dashed curve throughout Figs. 4(p) to 4(t)]. We mustnote that, at high illumination level [Fig. 4(p)], thebead detection dynamic range is limited by the para-

Fig. 2. Squared amplitude holograms �E�2 in logarithmicarbitrary units. (a) 200 nm beads, (b) 100 nm beads, and (c)50 nm beads. Scale bar is 10 �m. Horizontal profile tracesof �E�2 at the beads positions, averaged over 4 pixels [(d)–

(f)], in linear arbitrary units.

sitic light background (arrow 1), which is several or-ders of magnitude larger than the holographic-detection noise floor (arrow 2), obtained in a cut fromthe off-axis quietest region of the reconstructed im-age, away from the beads region. The noise floor[about 1010 A.U. on Fig. 4(p)], which is related to shotnoise of the LO beam, gives an absolute calibration ofthe bead peak [5�1014 A.U. on Fig. 4(k)], since it cor-responds to one photoelectron per pixel [12]. Withoutattenuation [Fig. 4(k)], the bead peak area is about20�20 pixels [see Fig. 4(p)]; it corresponds thus to�2�107e. 1 W of laser yields �1019 photons/s. With10 mW and 2�E=100 ms (two frames to make an ho-logram), we get 1016 photons. The Mie scatteringcross section of 200 nm gold particles (refractive in-dex; n=0.39−2.38j at 532 nm, with j2=−1) [17] is0.1 �m2. The total illumination area is �1 mm2; thuswe get 109 scattered photons per bead. Since the beadis located at an air–glass interface, most of the light

Fig. 3. �E�2 reconstructed images of a 200 nm gold beadwith experimental setup of Fig. 1(b) at several axial posi-tions: (a) z=22 �m, (b) z=17 �m, (c) z=0 �m, displayed inlogarithmic arbitrary units. Scale bar is 5 �m. Transverseplane profile traces of �E�2 at the beads positions, averagedover 3 pixels are shown in (d)–(f), in linear arbitrary units.Axial plane distribution (g) and linear scale profile aver-aged over 10 pixels in the lateral direction (h) are alsoshown.

Fig. 4. Intensity images calculated from 4�2 cameraframes data of a pair of 200 nm particles. (a)–(e) Direct im-age (LO beam is off), (k)–(o) holography (LO beam is on).Scale bar is 5 �m. Illumination field power attenuationranges from 100 to 104. Bead profiles without [(f)–(j)], andwith [(p) to (t)] LO beam. The vertical axis range (A.U.) is

the same for (f)–(j) and for (p)–(t).

502 OPTICS LETTERS / Vol. 33, No. 5 / March 1, 2008

�85% � is scattered within the glass [18], and 1.5�108 photons are scattered forward within a 2� solidangle. The collection solid angle of a NA=0.9 objec-tive is �� /4. We then get 4�107 photons on theCCD, yielding an expected signal of 2�107e (50%quantum efficiency at 532 nm), in agreement withthe experiment.

To assess imaging performances in experimentalconditions compatible with biological microscopy, wehave performed axial sectioning of dynamic beads;we have imaged a suspension of 200 nm beads inBrownian motion in water [see Fig. 1(c)]. A�30-�m-thick layer sample is realized within a slide/Parafilm/coverslip stack. The Parafilm layer washeated until melting to serve as waterproof spacer of�30 �m thickness. In this configuration, the top in-terface (coverslip–air) is the one where TIR occurs.The part of the illumination field EI not diffracted bythe beads undergoes TIR, while the scattered field Epasses through the MO and is used for imaging.Since the exposure time is short ��E=1 ms�, the LOpower is increased to fill the CCD dynamic range.Over a time �, Brownian particles travel by a dis-tance r���= �6DB��1/2, where DB=2.1�10−12 m2 s−1 isthe diffusion coefficient of a 200 nm particle in water.During one exposure, this travel is smaller than theparticle size r�1 ms��110 nm. 200 nm beads thus ap-pear to be quasi-immobile but travel from one imageto the next by r�80 ms��1 �m. This property is usedfor signal demodulation. The hologram is obtained bysubtracting from the current recorded frame the timeaverage of the ten next consecutive frames. In thisway the holographic information is recorded in oneexposure �E=1 ms. To localize smaller beads, the ex-posure time might be shortened accordingly.

Figure 5 shows a series of images [(a)–(d)] and pro-files [(e)–(h)] calculated from a single hologram. Im-ages (a)–(d) and profiles (e)–(h) are calculated ataxial positions z [(a) and (e)], z+10 �m [(b) and (f)],. . .z+30 �m [(d) and (h)]. A composite image (i) is

Fig. 5. (Multimedia online) Axial exploration of a 200 nmgold beads suspension in water. (a)–(d) �E�2 images recon-structed 10 �m apart. (e)–(h) Linear scale profiles averagedover 4 pixels, (i) composite image; scale bar is 5 �m.

made from reconstructed intensity holograms at

axial positions �z� coded in color in the 30 �m range.The attached multimedia file shows the video ratemotion of the particles in this composite image.

In this Letter we have performed full-field imagingof gold nanoparticles in 3D at videorate with expo-sure times as short as 1 ms. Our holographic tech-nique, which benefits from heterodyne gain, exhibitsoptimal detection sensitivity and, since the signal canbe easily calibrated, we have verified its agreementwith the expected signal. In the future, better axialresolution might be obtained by using the phase ofthe holographic data. Low-coherence light sourcesmight be used to reduce the background light compo-nent and improve depth sectioning. The backgroundmight also be reduced by a selective modulation (e.g.,spatial or photothermal) of the particles.

The authors acknowledge support from the FrenchAgence Nationale de la Recherche (ANR) and theCentre de compétence NanoSciences Île de France(C’nano IdF).

References

1. S. Schultz, D. R. Smith, J. J. Mock, and D. A. Schultz,Proc. Natl. Acad. Sci. USA 97, 996 (2000).

2. C. Sonnichsen, S. Geier, N. E. Hecker, G. von Plessen,J. Feldmann, H. Ditlbacher, B. Lamprecht, J. R.Krenn, F. R. Aussenegg, V. Z.-H. Chan, J. P. Spatz, andM. Möller, Appl. Phys. Lett. 77, 2949 (2000).

3. D. Boyer, P. Tamarat, A. Maali, B. Lounis, and M.Orrit, Science 297, 1160 (2002).

4. K. Lindfors, T. Kalkbrenner, P. Stoller, and V.Sandoghdar, Phys. Rev. Lett. 93, 037401 (2004).

5. M. A. van Dijk, M. Lippitz, and M. Orrit, Phys. Rev.Lett. 95, 267406 (2005).

6. F. V. Ignatovich and L. Novotny, Phys. Rev. Lett. 96,013901 (2006).

7. V. Jacobsen, P. Stoller, C. Brunner, V. Vogel, and V.Sandoghdar, Opt. Express 14, 405 (2006).

8. J. S. Batchelder and M. A. Taubenblatt, Appl. Phys.Lett. 55, 215 (1989).

9. J. Hwang and W. E. Moerner, Opt. Commun. 280, 487(2007).

10. S. Berciaud, D. Lasne, G. A. Blab, L. Cognet, and B.Lounis, Phys. Rev. B 73, 045424 (2006).

11. M. Atlan, M. Gross, and E. Absil, Opt. Lett. 32, 1456(2007).

12. M. Gross and M. Atlan, Opt. Lett. 32, 909 (2007).13. W. Lukosz and R. E. Kunz, J. Opt. Soc. Am. 71, 744

(1981)14. M. Speidel, A. Jon, and E. L. Florin, Opt. Lett. 28, 69

(2003).15. D. Patra, I. Gregor, J. Enderlein, and M. Sauer, Appl.

Phys. Lett. 87, 101103 (2005).16. J. Mertz, J. Opt. Soc. Am. B 17, 1906 (2000).17. http://omlc.ogi.edu/calc/mie-calc.html.18. X. Brokmann, E. Giacobino, M. Dahan, and J. P.

Hermier, Appl. Phys. Lett. 85, 712 (2004).