Supporting Information. Single-Particle Absorption Spectroscopy by. Photothermal Contrast
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1 Supporting Information Single-Particle Absorption Spectroscopy by Photothermal Contrast Mustafa Yorulmaz 1,, Sara Nizzero 2, 3,, Anneli Hoggard 1, Lin-Yung Wang 1, Yi-Yu Cai 1, Man-Nung Su 1, Wei-Shun Chang 1, Stephan Link 1, 3, * 1 Department of Chemistry, 2 Applied Physics Graduate Program, 3 Department of Electrical and Computer Engineering, Laboratory for Nanophotonics, Rice University, Houston, Texas 77005, United States These authors contributed equally to this work * Corresponding author s slink@rice.edu S1
2 Content 1.) Single-particle absorption spectroscopy 2.) Sample preparation 3.) Size distribution of nanoparticles with nominal diameters of 20 nm and 50 nm 4.) Photothermal time trace to record the absorption spectrum 5.) FDTD calculations 6.) Gold film thickness measured by AFM 7.) Pointing stability 8.) Material independence of the calibration procedure 9.) Measured film spectrum at different spatial positions 10.) Corrected nanoparticle spectrum using different excitation angles 11.) Power dependence of the nanoparticle absorption spectrum 12.) Power dependence of the photothermal signal at different wavelengths 13.) Hyperspectral scattering spectroscopy and spatial correlation between absorption and scattering spectroscopy 14.) Microscope stability 15.) Reproducibility of the measured absorption spectrum 16.) Size distribution of nanorods S2
3 1.) Single-particle absorption spectroscopy Single-particle photothermal spectroscopy was performed in transmission mode on a home-built microscope constructed around a commercial microscope (Zeiss Axia Observer D1). Differently from the previously reported photothermal microscopy, which employs a cw excitation laser, here we used a pulsed supercontinuum laser source (Fianium, Whitelase SC450, 6 W, 60 MHz, 12 ps) equipped an AOTF. The laser was linearly polarized with an anisotropy of 97% before entering the microscope, unless otherwise stated. The wavelength selection and the intensity modulation at 230 khz of the heating beam was made using the AOTF, which was driven by a frequency generator (FLUKE MHz DDS). The probe beam was provided by a cw HeNe laser (Melles Griot 25-LHP ) with a wavelength of 633 nm or a cw 785 nm laser (Coherent, OBIS 785 LX). The probe beam was overlapped with the heating beam on a dichroic mirror (Chroma Technology Corp. ZQ633RDC) or a beam splitter (R/T 50/50, Chroma) and the axial overlap of the two beams was adjusted with the help of a telescope system. Both beams were directed onto the sample through a microscope objective (Zeiss, numerical aperture (N.A.) = 1.4, 63X, oil, Plan Apochromat). The energy absorbed by the nanoparticles by the excitation beam was released as heat in the vicinity of the nanoparticle surfaces leading to a time dependent refractive index change, i.e. a thermal lens, caused by the intensity modulation of the heating laser. The probe laser focused to the same spot was scattered by this thermal lens. A photothermal beat note signal was created by the interference of the scattered probe field with the reference probe field, which in this case was the transmitted probe laser. The interference signal was collected by another microscope objective (Zeiss, N.A. = 0.6, 40X, air, LD Achroplan) in a transmission geometry and detected by a Si photoreceiver (FEMTO HCA-S 200M-Si) connected to a variable gain amplifier (FEMTO DHPCA-100). The output signal was fed into a lock-in amplifier (Stanford Research Systems S3
4 SR-844) with a reference frequency provided by the function generator. For the experiments with cw laser excitation, the pulsed laser and the AOTF were replaced with a 532 nm cw laser (Coherent Verdi V6) and an acousto-optic modulator (IntraAction AOM-402AF1), while the rest of the setup remained the same. The experiments were controlled by using a home-written LabVIEW program. Photothermal images were acquired by raster scanning the sample across the focused lasers using a three-axis piezo scanning stage (Physik Instrumente P-157.3CL). For absorption spectroscopy, single gold nanoparticles were brought to the position of the focused lasers and the signal was collected as a function of time, while the wavelength was changed continuously each 10 s in steps of 10 nm (each 5 s in steps of 5 nm for absorption spectra of nanorods) using the software provided. Conversion of time transients into spectra was accomplished by averaging the signals for each 6 8 s (or 3 s) time bin for a certain wavelength, considering the 1 s response time of the AOTF. The variation of the signal around the mean value was less than 2%. The order of the measurement between nanoparticle and film had no effect on the final nanoparticle spectrum. We also intentionally magnified the heating beam waist by 3 times to overfill the back aperture of the objective to reduce the dimensions of the photothermal profile, but were unable to obtain smaller values than given in Fig. 2. S4
5 2.) Sample preparation Figure S1. Schematic representation of the sample geometry. The embedding medium surrounds the nanoparticles and metal film, and it is confined by the spacer and the top glass slide. Gold nanoparticles were immobilized on the surface of an indexed cover slip as illustrated in Fig. S1. The gold pattern was used for correlated absorption and scattering measurements and for performing the correction procedure described in the main text. A uniform distribution of nanoparticles with interparticle distances larger than 1 µm was obtained by spincoating, enabling measurements at the individual nanoparticle level. The gold film was designed to be 15 nm thick and was deposited on the cover slip via evaporation of metal (Au or Al) through a TEM mask. Prior to nanoparticle deposition, the substrates were cleaned with a 1:4:20 solution of NH 4 OH:H 2 O 2 :H 2 O. A 2 nm Ti film was deposited first to facilitate the adhesion of the gold film. We independently measured the thickness of the gold film using a spectroscopic imaging ellipsometer (Nanofilm, EP 3 ) for substrates used for experiments performed on spherical nanoparticles and found it to be 13±1 nm, in good agreement with the expected value. For experiments performed on nanorods, we prepared a new batch of patterned substrates and verified the thickness of the gold film using AFM as shown in Fig. S5. The metal indexed substrate was then plasma-cleaned and functionalized using Vectabond TM in acetone for the samples with spherical nanoparticles. Vectabond TM was used to create amine group termination on the substrate S5
6 surface, and thereby increased the adhesion of the spherical nanoparticles. For the samples with nanorods, the indexed substrate was used without additional changes to the surface chemistry. An aqueous solution of nanoparticles was spin-coated for 60 s at 2,500 rpm enabling a homogeneous distribution of nanoparticles on the surface. Finally, a few drops of embedding solvent (water, glycerol, or oil) were deposited on top of the sample, which was sealed with the help of a spacer (Grace TM Bio-Labs, depth = 500 µm) and a clean glass cover slip. S6
7 3.) Size distribution of nanoparticles with nominal diameters of 20 nm and 50 nm. Figure S2. Transmission electron microscopy (TEM) to reveal the distribution of sizes in the nanoparticle suspensions. a, TEM image of nominal 20 nm diameter particles. b, TEM image of nominal 50 nm diameter particles. Distribution of nanoparticle diameters with nominal sizes of c, 20 nm and d, 50 nm. Fig. S2 shows that the average diameters of nanoparticles with nominal sizes of 20 nm and 50 nm were 19 ±2 nm and 49 ± 4 nm, respectively. The samples were purchased from BBInternational. S7
8 4.) Photothermal time trace to record the absorption spectrum Figure S3. Principle of single-particle absorption spectroscopy. Photothermal signal as a function of time measured on the gold particle (black), gold film (blue), and background (red). These time traces were taken with an average power of 30 µw at λ exc = 550 nm. In order to retrieve the absorption spectrum of individual nanoparticles, we recorded the photothermal signals of nanoparticles while we were changing the wavelength after each 10 s time interval as shown in Fig. S3. For spherical nanoparticles, the wavelength was gradually varied from 470 nm to 700 nm with 10 nm step sizes. Therefore, the recorded time traces were 240 s long. The signal that was acquired for times between 150 s and 180 s corresponded to the measurement performed at 620 nm, 630 nm, and 640 nm, respectively. At 620 nm and 640 nm, the nanospheres and the film did not show a signal higher than the background due to the very low heating power that reached the sample, a consequence of using the 633 nm dichroic mirror in the excitation path to combine the heating and the probe lasers. All signals at 630 nm were even smaller than the background at 620 nm and 640 nm because the detector saturated at this wavelength despite the presence of a 633 nm bandpass filter in the detection path to select the signal at the probe laser wavelength as part of the 630 nm heating laser power passed the filter. Therefore, the data for this wavelength was always excluded from the spectra of nanospheres. For measurements on nanorods, there was no such limitation because a 785 nm probe laser with matching bandpass filters in the detection path of the photothermal microscope was used. S8
9 5.) FDTD calculations Figure S4. FDTD simulation geometry, the effect of the medium on the absorption spectrum of a gold film, and comparison of measured and simulated transmission of the thin film a, Schematic illustration of the FDTD simulations using a commercially available FDTD package 1. The wave vector k is normal to the substrate. The red box represents the discrete Fourier transform (DFT) monitors, which record the normalized transmission (T) and reflection (R) power. The perfect matching layer (PML) defines the total simulation volume (400 nm 600 nm 600 nm). The optical constants of gold and of Ti were adopted from the experimental values 2,3. A Gaussian beam with a waist of 350 nm was used for the excitation of the film. The thickness of Ti and gold layers used in this simulation were 2 nm and 15 nm, respectively. b, Calculated normalized absorption spectra (1-(R+T)) of a gold film in different media. c, Calculated and measured transmission efficiencies of a gold film. The simulation was performed for a sample with a 2 nm Ti layer and 9 nm gold layer. The total height was obtained using AFM as shown in Fig. S5. The absorption spectrum of a thin gold film was calculated using a commercially available finite-difference-time-domain (FDTD) package 1. The model geometry is shown in Fig. S4a. A Gaussian beam with a waist of 350 nm was used for excitation of the film in the simulation. The energy-dependent optical constants of gold were adopted from the tabulated values for bulk gold measured by Johnson and Christy 2. The optical constants of Ti were also adopted from the experimental values 4. The glass substrate in the experiment was modeled as a SiO 2 dielectric S9
10 layer using a constant refractive index of The grid size used in this simulation was 2 nm. A default convergence criterion was used. The calculations were carried out using the Shared Tightly-Integrated Cluster (STIC) provided by Rice University. Each job required totally 36 CPUs with approximately one-hour run time. The wavelength dependent absorption spectra were obtained by subtracting the transmission spectrum (T) and reflection spectrum (R) from unity, i.e. 1-(R+T). Calculated film spectra in different media, glycerol and water, are shown in Fig. S4b. We compare the absolute value of transmission efficiency and the lineshape of measured and simulated transmission spectra in Fig. S4c. We observe a good agreement between theory and experiment. Therefore, we conclude that we can use the absorption spectrum obtained by using FDTD simulations. S10
11 6.) Gold film thickness measured by AFM Figure S5. Thickness of gold film on glass substrate used for absorption spectroscopy of single gold nanorods measured by atomic force microscopy in tapping mode. a, AFM height image shows bright and dark signal areas that correspond to heights of the gold film including Ti adhesion layer and glass, respectively. b, The height along the blue line drawn in a is shown. We found the average thickness to be as 10.9 ± 0.3 nm by averaging the height over a few microns in the line profile. The thickness of the Ti layer was 2 nm, and therefore the gold film thickness was ~ 9 nm. S11
12 7.) Pointing stability Figure S6. Normalized absorption spectra of the same nanorod obtained by optimizing the signal at different wavelengths. We checked the spatial pointing stability of the laser by plotting the spectrum of the same nanorod after optimizing the signal at different excitation wavelengths by moving the nanoparticle with respect to the beam. Each time the signal was optimized on the nanoparticle, then the measurements on the nanoparticle and the film were performed under the same conditions. The plotted spectra do not show a significant deviation from each other. Therefore we rule out the possibility of spatial laser instability. S12
13 8.) Material independence of the calibration procedure Figure S7. Independence of the film material on the calibration procedure. Absorption spectra of two different nanoparticles (nominal size 50 nm) calibrated with thin films of different materials, i.e. gold and aluminum. The blue squares correspond to a measurement with a calibration factor obtained from a 2 nm titanium + 15 nm gold film, while the red squares correspond to a measurement where the calibration was done with a 15 nm aluminum film. The material of the thin film used for the calibration did not affect the success of the correction as shown in Fig. S7. Corrections carried out with gold or aluminum films generated absorption spectra that are in agreement with the simulated absorption spectrum of a 44 nm diameter gold nanoparticle. Thus, our calibration method is independent of the film material. S13
14 9.) Measured film spectrum at different spatial positions Figure S8. Film spectra at different positions on the gold film and resulting corrected nanoparticle spectra. a, The two measured spectra were measured at different locations of the gold film. b, The corrected spectrum of the same nanoparticle using the two film spectra measured at different spatial positions. We did not observe any significant difference in the measured absorption spectra of the thin film at distant locations on the same sample. Moreover, the corrected absorption spectra of the same nanoparticle using different film spectra were virtually identical to each other. Therefore, we can conclude that the film was homogeneous with little local variations including its thickness. The correction of a nanoparticle spectrum was therefore independent of the position where the film absorption spectrum was measured. S14
15 10.) Corrected nanoparticle spectrum using different excitation angles Figure S9. Effect of the excitation angle on the measured film spectrum and the corrected nanoparticle spectrum. a, Measured film spectrum under different excitation conditions, i.e. using objectives with numerical apertures (N.A.) of 0.7 and 1.4. b, Absorption spectra of the same nanoparticle obtained by performing the film and nanoparticle measurements with the low and high numerical aperture objectives, respectively. The gold film spectra measured under various excitation angles via the use of different objectives were very similar to each other (Fig. S9a), in particular with respect to the spectral shape. The measured film spectra were then used to correct the same nanoparticle spectrum measured using the corresponding numerical aperture objective. The corrected absorption spectra of the same nanoparticle were almost identical to each other (Fig. S9b). Therefore, we conclude that the thermal lens geometry does not have an influence on the lineshape of the corrected absorption spectrum of the nanoparticle. S15
16 11.) Power dependence of the nanoparticle absorption spectrum Figure S10. No significant broadening due to damping of the nanoparticle plasmon upon heating. The absorption spectrum of the same nanoparticle was measured at three heating powers to check for linewidth broadening due to increased heating. We observe minimal variations in the measured absorption spectra upon using different heating powers as illustrated in Fig. S10. In particular, we did not observe any significant broadening of the absorption spectrum. S16
17 12.) Power dependence of the photothermal signal at different wavelengths Figure S11. Power dependence of photothermal signal at different wavelengths. a, Calculated absorption spectrum of a spherical nanoparticle (radius = 24 nm, n = 1.495) using Mie theory. Points highlighted on the spectrum show the wavelengths at which the power dependence of photothermal signal under pulsed excitation was tested. b, Measured power dependence of the photothermal signal at different wavelengths. All trends are linear over the entire power range tested. The photothermal signal changes linearly with excitation power at different wavelengths around the plasmon resonance as shown in Fig. S11. This data confirms that the measured signal is linear in nature and that no spectral broadening occurs. S17
18 13.) Hyperspectral scattering spectroscopy and spatial correlation between absorption and scattering spectroscopy Figure S12. Correlation of absorption and scattering signals. Spatial correlation of nanoparticles selected in the a, DSLR image (Canon, Rebel EOS T2i) were measured in b, absorption and in c, scattering. The use of an indexed film allowed the localization of a specific area and thus the spatial correlation between different measurements. All these images refer to a sample of 50 nm diameter gold nanoparticles deposited onto a glass coverslip. The white square box in a and the images in b and c are 20 µm 20 µm. Conventional dark-field spectroscopy in combination with an automated hyperspectral imaging scheme built around a commercial microscope (Zeiss Axia Observer D1) was used for recording S18
19 the scattering spectra of individual nanoparticles. 4 Single nanoparticles were excited using a dark-field condenser (Zeiss, illumination N.A. was between 1.2 and 1.4). A 3-dimensional data cube, which contained spatial location, intensity, and the spectra of the corresponding pixel, was recorded by moving the slit of the spectrograph (Princeton Instruments Acton SpectraPro 2150i) equipped with a thermoelectrically cooled back-illuminated CCD camera (Princeton Instruments PIXIS) with respect to the field of view of the microscope objective (Zeiss, variable N.A. between 0.7 and 1.4, 63X, oil, Plan Apochromat). The experiment was controlled and data was collected using a home-written LabVIEW program. The scattering spectrum for each nanoparticle was obtained by subtracting the residual background from the nanoparticle signal and accounting for the spectral response of the excitation lamp and the CCD camera. We used indexed gold films evaporated on cover slips which allowed recognition of the scanned area and enabled correlating the absorption and scattering images and spectra of the same individual nanoparticles. Note that the same film was used for the correction procedure as well. For correlated absorption and scattering spectroscopy of the same nanoparticle, scattering measurements were typically carried out first. After the absorption spectroscopy, scattering spectroscopy was repeated on the same nanoparticles to rule out photothermal reshaping. For spatial correlation, a picture was first taken with a DSLR camera in the dark-field microscopy configuration (Fig. S12a). The color of the round spots in this picture gave an idea about the shape of the nanoparticles. The edges of the gold film showed an intense orange color. Absorption image was taken with an excitation wavelength of 532 nm (Fig. S12b). Scattering spectra were taken in the hyperspectral microscope (Fig. S12c). S19
20 14.) Microscope stability Figure S13. Stability of the photothermal signal at different wavelengths. The photothermal signal of a 50 nm diameter gold nanoparticle in glycerol as a function of time. The measurement was performed at different wavelengths. The stability of the photothermal signal during the time period of the spectral acquisition is important to ensure the reliability on the measurement. In Fig. S13 we show that the stability for the time required to measure single-particle absorption spectra was very good. S20
21 15.) Reproducibility of the measured absorption spectrum. Figure S14. Error in the measured absorption spectra for different powers. The relative variations in the photothermal signal of 20 nm diameter nanoparticles embedded in glycerol as a function of wavelength for different heating powers. In order to obtain the relative error, the spectrum of the same nanoparticle was measured 5 times for each average power of 30 µw, 40 µw, and 50 µw (at 550 nm). Then, the spectra were averaged and the mean values and the standard deviations were obtained. Each data point corresponds to the standard deviation normalized by the average signal. The dashed-dotted line indicates 20% measurement error. The reproducibility of the photothermal absorption spectroscopy experiments was very good with most of the data points having an error less than 20% (dash-dotted line in Fig. S14), and significantly lower errors observed for wavelengths between 520 nm and 600 nm. The data in Fig. S14 was obtained for 20 nm diameter gold nanoparticles which were embedded in glycerol and immobilized on a glass substrate. S21
22 16.) Size distribution of nanorods Figure S15. Transmission electron microscopy (TEM) of small-volume nanorods TEM image of nanorods with nominal widths, lengths, and aspect ratios of 22±3 nm, 49±6 nm, and 2.4±0.4, respectively. S22
23 Figure S16. Transmission electron microscopy (TEM) of large-volume nanorods TEM image of nanorods with nominal widths, lengths, and aspect ratios of 41±4 nm, 60±6 nm, and 1.5±0.2, respectively. S23
24 References (1) FDTD Solutions v ; Lumerical Solutions, Inc: Vancouver, Canada, 2013 (2) Johnson, P. B. and Christy, R. W. Optical constants of the noble metals. Phys. Rev. B 1972, 6, (3) Palik, E. D. (ed.), Handbook of Optical Constants of Solids, Academic Press, Orlando, 1985 (4) Byers, C. P., et al. Single-particle spectroscopy reveals heterogeneity in electrochemical tuning of the localized surface plasmon. J. Phys. Chem. B 2014, 118, S24
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