Photothermal Heterodyne Imaging of Individual Nonfluorescent Nanoclusters and Nanocrystals Stephane Berciaud, Laurent Cognet, Gerhard A. Blab, and Brahim Lounis Centre de Physique Moleculaire Optique et Hertzienne, CNRS et Universite Bordeaux Ayca Yalcin

Outline Motivation Previous Technique Method of Detection Results and Applications Conclusion

Using scattering properties Fluorescent molecules: –Photobleaching  short observation times Semiconductor nanoparticles: –More stable, but blinking (On longer time scales (t>100 ns), a semiconductor nanocrystal presents a succession of time intervals during which it emits light ("on-state") or not ("off-state"). –Difficult to functionalize

Using absorptive properties Large absorption cross section Short time interval between successive absorption events  Metal nanoparticles - excited near their plasmon resonance, large absorption cross section (~8x cm 2 for a 5nm diameter particle) - fast electron-phonon relaxation time (in the ps range). - luminescence is extremely weak, almost all the absorbed energy is converted into heat. - temperature rise induced by the heating leads to a variation of the local index of refraction.

PIC (Boyer et al.) Heating beam modulated at high frequency induces periodic phase difference between two red beams, which gives rise to modulation of detected red intensity. 5nm particles

Effect of scattering on Photothermal Image 300nm latex spheres, 80nm/10nm gold spheres –A: differential interference contrast –B: photothermal (30kW/cm 2 ) –C: photothermal (1.5MW/cm 2 )

Photothermal Effect When a gold nanoparticle in a homogeneous medium is illuminated with an intensity modulated laser beam, it behaves like a heat point source with a heating power: Ω: modulation frequency P heat: the average absorbed laser power It generates a time-modulated index of refraction in the vicinity of the particle with a spatiotemporal profile: r: distance from particle n: index of refraction R th : characteristic length for heat diffusion K: thermal conductivity C: heat capacity per unit volume

SETUP (red) probe beam (720 nm, single frequency Ti:sapphire laser) (green) heating beam (532 nm, frequency doubled Nd:YAG laser) intensity modulated at (100 kHz to 15 MHz) by an acousto-optic modulator. high aperture objective (100X, Zeiss, NA=1.4) power of the heating beam: from less than 1 μW to 3.5 mW at the objective. Experimental Setup

Sample Preparation Samples prepared by spin coating a solution of gold nanoparticles [diameter of 1.4, 2, 5, 10, 20, 33, or 75nm, diluted into a polyvinyl-alcohol (PVOH) matrix, 2% mass] onto clean microscope coverslips. The dilution and spinning speed were chosen such that the final density of spheres in the sample was less than 1 μm -2. Application of a silicon oil on the sample ensures homogeneity of the heat diffusion. The size distribution of the nanospheres was checked by transmission electron microscopy and was in agreement with the manufacturer’s specification.

Some Results 3D representation of a photothermal heterodyne image of gold aggregates of 67 atoms (1.4 nm diameter nanogold). Relatively small heating power (~3.5 mW) and a remarkably large signal-to-noise ratio (SNR > 10), shot noise limited. Confirmed that the peaks stem from single particles by generating the histogram of the signal height for 272 imaged peaks. Monomodal distribution with a width in agreement with the spread in particle size.

Comparison with Theory In order to estimate the measured signal, theory of ‘‘scattering from a fluctuating dielectric medium’’ is used to calculate the field scattered by the modulated index profile. The beating between the reference and scattered fields leads to a beating power S at the detector:  : geometry factor close to unity I inc : incident red intensity at the particle location P ref : reference (backreflected) beam power f, g: two dimensionless functions which depend on the modulation frequency and the thermal diffusivity of the medium

Measured Signal vs. Frequency The variations o f κ ( Ω)/Ω and g κ ( Ω)/Ω for κ/C=2x10 -8 m 2 /s. At low frequencies, R th is larger than the probe spot size (~λ/2) and f κ ( Ω)/Ω is preponderant. At high frequencies, R th << λ, g κ ( Ω)/Ω dominates and decreases as 1/ Ω. The magnitude of demodulated signal delivered by the lock-in amplifier is proportional to The frequency dependence of this signal measured on single particles (5nm) is shown.

Measured Signal vs. Heating Power Single 2nm gold nanoparticle, σ~5x cm 532 nm, P heat =10nW for 2MW/cm 2 illumination. P inc =70mW, Ω/2 π =800kHz, P ref =100μW  Calculated S dem =5nW.  Measured: ~2nW. Linear dependence of the signal with heating power. No saturation for increased power, however, fluctuations in the signal amplitude and eventually irreversible damage on the particle.

Application Size dependence of absorption cross section of gold 532nm with diameter nm. For each sample, histogram of signal amplitudes is generated. Histograms displayed bimodal distributions, the mean of each population was measured, and size dependence of the absorption cross section normalized to that of 10 nm particles is obtained.

Luminescent semiconductor nanocrystals or fluorescent molecules have radiative relaxation times in ns range, difficult to detect by absorption. At high excitation intensities, efficient nonradiative relaxation pathways appear. Fluorescent image of CdSe/ZnS quantum dots (peak emission at 640 nm) excited by the heating beam at 0.1 kW/cm 2, blinking behavior visible. Photothermal heterodyne image at 5kW/cm 2 where quantum dots are no longer luminescent. Two images correlate well, ensuring spots are individual quantum dots (>90% of the fluorescent spots correlate with a photothermal spot). No blinking behavior, initially non- fluorescent quantum dots are now detected. Luminescence vs. Photothermal

Biological Applications In the current configuration, a 5nm gold nanoparticle can be detected with a SNR>100 at a heating power of 1mW. At this power, a local temperature increase of 4K is estimated in aqueous solutions. As the temperature reduces as the inverse of distance, and most microscopy applications in biosciences do not require such a high SNR, imaging is possible by inducing a local heating of far less than 1K above the average temperature in the sample.

Conclusion Advantages of photothermal heterodyne detection –no photobleaching or blinking –Immune to background effects SNR>10 for 1.4nm gold particles, first detection As any far-field optical technique, it has wavelength limited resolution –combine with near-field optical techniques  sensitivity+resolution Can apply to many diffusion and colocalization problems in physical chemistry and material science and to track labeled biomolecules in cells.