This work is supported by Ministry of Science and Technology under NSC-101-2923-M-002-001-MY3 and NSC-102-2112-M-002-018-MY3. This research is also supported by the Japan Society for the Promotion of Science (JSPS) through the &#8220;Funding Program for Next Generation World-Leading Researchers (NEXT Program),&#8221; initiated by the Council for Science and Technology Policy (CSTP) and JSPS Asian CORE Program.

1. GNS Sample Preparation
<ol>
	<li>Before preparing the sample, sonicate 1 ml GNS colloid solution for at least 15 min at about 40 kHz to prevent particle aggregation, which may cause the LSPR peak to shift.</li>
	<li>Drop 100-200 &#181;l of GNS colloid onto a slide glass with commercial magnesium aluminum silicate (MAS) coating to fix the GNSs.</li>
	<li>After at least 1 min, remove the extra colloid by flushing with distilled water. The waiting time depends on the required distribution density of the GNSs. Typically...

<ol>
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The authors declare that they have no competing financial interests.

In the protocol, there are several critical steps. First, when preparing the samples, the density of nanoparticles should not be too high, to avoid plasmonic coupling among particles. If two or more particles are very close to each other, the coupling results in the LSPR wavelength shifting toward longer wavelengths, thus significantly reducing the nonlinearity. However, this imaging technique actually maps the distribution of plasmonic modes, instead of the particles themselves. Therefore, it is expected that with an ap...

The study of plasmonics has attracted great interest due to its applications in many different fields1-4. One of the most investigated fields in plasmonics is surface plasmonics, in which the collective oscillation of conduction electrons couples with an external electromagnetic wave at an interface between a metal and dielectric. Surface plasmonics has been explored for its potential applications in subwavelength optics, biophotonics, and microscopy5,6. The strong field enhancement in the ultra-small volume of metallic nanoparticles due to localized surface plasmon resonance (LSPR) has attracted extensive attention, not only because of its exceptional sensitivity to particle sizes, particle shapes, and the dielectric properties of the surrounding medium7-10, but also because of its ability to boost inherently weak nonlinear optical effects11. The exceptional sensitivity of LSPR is valuable for bio-sensing and near-field imaging techniques12,13. On the other hand, the enhanced nonlinearity of plasmonic structures can be utilized in photonic integrated circuits in applications such as optical switching and all-optical signal processing14,15. It is well known that the plasmonic absorption is linearly proportional to the excitation intensity at low intensity levels. When the excitation is strong enough, the absorption reaches saturation. Intriguingly, at higher intensities, the absorption increases again. These nonlinear effects are called saturable absorption (SA)15-17 and reverse saturable absorption (RSA)18, respectively.
It is known that due to the LSPR, scattering is particularly strong in plasmonic structures. Based on fundamental electromagnetics, the response of scattering versus incident intensity should be linear. However, in nanoparticles, scattering and absorption are closely linked via the Mie theory, and both can be expressed in terms of real and imaginary parts of the dielectric constant. Under the assumption that a single GNS behaves as a dipole under light illumination, the scattering coefficient (Qsca) and absorption coefficient (Qabs) from a single plasmonic nanoparticle according to the Mie theory can be expressed as19
<img alt="Equation 1" src="/files/ftp_upload/53338/53338eq1.jpg" />
where x is 2&#960;a/&#955;, a is the radius of the sphere, and m2 is &#949;m/&#949;d. Here, &#949;m and &#949;d correspond to the dielectric constants of the metal and of the surrounding dielectrics, respectively. Since the form of the scattering coefficient is similar to that of the absorption coefficient, it is therefore expected to observe saturable scattering in a single plasmonic nanoparticle20.
Recently, nonlinear saturable scattering in an isolated plasmonic particle was demonstrated for the first time21. It is remarkable that at deep saturation, the scattering intensity in fact decreased slightly when the excitation intensity increased. Even more remarkably, when the excitation intensity continued increasing after the scattering became saturated, the scattering intensity rose again, showing the effect of reverse saturable scattering20. Wavelength- and size-dependent studies have shown a strong relationship between LSPR and nonlinear scattering21. The intensity and wavelength dependences of plasmonic scattering are very similar to those of absorption, suggesting a common mechanism underlying these nonlinear behaviors.
In terms of applications, it is well known that nonlinearity helps to improve optical microscopy resolution. In 2007, saturated excitation (SAX) microscopy was proposed, which can enhance resolution by extracting the saturated signal via a temporal sinusoidal modulation of the excitation beam22. SAX microscopy is based on the concept that, for a laser focal spot, the intensity is stronger at the center than at the periphery. If the signal (either fluorescence or scattering) exhibits saturation behavior, the saturation must start from the center, while the linear response remains at the periphery. Therefore, if there is a method to extract only the saturated part, it will leave only the central part while rejecting the peripheral part, thus effectively enhancing the spatial resolution. In principle, there is no lower resolution limit in SAX microscopy, as long as deep saturation is reached and there is no sample damage due to the intense illumination.
It has been shown that the resolution of fluorescence imaging can be significantly enhanced by utilizing the SAX technique. However, fluorescence suffers from the photobleaching effect. Combining the discovery of scattering nonlinearity and the concept of SAX, super-resolution microscopy based on scattering can be realized21. Compared to conventional super-resolution microscopies, the scattering-based technique provides a novel non-bleaching contrast method. In this paper, a step-by-step description is given to outline the procedures required to obtain and extract the nonlinearity of plasmonic scattering. Methods of identifying scattering nonlinearities introduced by changing the incident intensity are described. More details will be provided to unravel how these nonlinearities affect images of single nanoparticles and how spatial resolution can be enhanced accordingly by the SAX technique.

<table>
	<tbody>
		<tr>
			<td>microscope body</td>
			<td>Olympus, Japan</td>
			<td>BX-51</td>
			<td></td>
		</tr>
		<tr>
			<td>objective lens</td>
			<td>Olympus, Japan</td>
			<td>UPlanSapo, 100X, NA 1.4</td>
			<td></td>
		</tr>
		<tr>
			<td>80-nm gold colloid</td>
			<td>BBI Solutions, UK</td>
			<td>EM.GC80</td>
			<td></td>
		</tr>
		<tr>
			<td>supercontinuum laser</td>
			<td>Fianium, United Kingdom</td>
			<td>SC400-2-PP</td>
			<td></td>
		</tr>
		<tr>
			<td>broadband dielectric mirrors</td>
			<td>Thorlabs, USA</td>
			<td>BB1-E02</td>
			<td></td>
		</tr>
		<tr>
			<td>field emission SEM</td>
			<td>JEOL, Japan</td>
			<td>JSM-6330F</td>
			<td>optional</td>
		</tr>
		<tr>
			<td>spectrometer</td>
			<td>Andor Technology, UK</td>
			<td>Shamrock 163</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;charge-coupled device</td>
			<td>Andor Technology, UK</td>
			<td>iDus DV420A-OE</td>
			<td></td>
		</tr>
		<tr>
			<td>acousto-optic modulators</td>
			<td>&#160;IntraAction Corp., USA</td>
			<td>AOM-402AF1</td>
			<td></td>
		</tr>
		<tr>
			<td>lock-in amplifier</td>
			<td>Stanford Research Systems, USA</td>
			<td>SR-830</td>
			<td></td>
		</tr>
		<tr>
			<td>MAS-coated slide glass</td>
			<td>Matsunami Glass, Japan,</td>
			<td>S9215</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of scattering nonlinearities from a single plasmonic nanoparticle

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. 
Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. 
In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Figure 6 shows the measured spectrum from an 80 nm GNS. A calculated curve based on the Mie theory is given in the same plot, showing excellent agreement. The LSPR peak is around 580 nm. In the following experiment, the laser wavelength was 532 nm, which was chosen as it is located inside the plasmonic band to enhance optical scattering with plasmonic effect and enable scattering saturation21.
Figure 7 presents scattering images of a single gold nan...

Watch this Scientific Journal Video about Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle at JoVE.com

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle

Saturable and reverse saturable scattering were discovered in isolated plasmonic particles and adopted as a novel non-bleaching contrast method in super-resolution microscopy. Here the experimental procedures of detecting and extracting nonlinear scattering are explained in detail, as well as how to enhance resolution with the aid of saturated excitation microscopy.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus ...

The overall goal of this experiment is to demonstrate resolution enhancement with non-linear scattering from plasmonic nanostructures. In the field of optical microscopy, contrast and the resolution are the most important factors. Our technique expand to the super resolution field with the new scattering based on contrast.The main advantage of this technique is that scattering from nanoparticles is strong, highly nonlinear, and non-bleaching. Demonstrating the procedure will be Hsuan Lee. A greater student from my lab.The experiment will be performed on an optical bench. Where there is a microscope and two laser sources. The first laser source is a 532 nanometer continuous wave laser used for imaging.The second is a super-continuum laser used for spectroscopy measurements. This is a schematic of the basic set up for both imaging and spectroscopy. Samples will be mounted on a piezoelectric positioning stage in the confocal microscope.Light from the 532 nanometer laser passes through a neutral density filter. The beam is directed by a beam splitter to galvano mirrors that provide raster scanning in the focal plane of the confocal microscope objective. The backward scattered light is collected by the objective and focused on the detector.Begin by aligning the white light illumination path of the microscope. Use the halogen light source under curler illumination. To verify the alignment of the internal iris, be certain to observe sharp edges on the octagon via the eyepiece or CCD.Next, use a piece of paper to check the external light path. Verify the beams are partially reflected by the beam splitter and propagate toward the laser by moving the paper along the light path. Now, have ready paper targets to aid in further alignment.The targets should be on thin paper with concentric rings. Place these targets along the beam path so that they are aligned with the halogen beam. Here, a target is in position on the back aperture of the objective lens.This is another target in position along the optical path. Next, turn on the 532 nanometer laser used for imaging in order to align it. Use the paper targets to aid in collimating the incident laser light opposite the halogen beam.Continue with finer alignment of the laser beam before removing the targets and proceeding. After aligning the laser, obtain a prepared gold nanosphere sample for the experiment. This sample has 80 nanometer gold nanospheres in oil.The mixture is sealed between two glass plates. Continue by mounting the sample on the microscope stage and adding objective oil. At this point, ready the detection system for imaging.Collect the scattered light with the photo multiplier tube at the end of the optical path. Back along the beam path, have a 20 micrometer diameter pinhole to block out of focus scattered light from the photo multiplier tube. Proceed with imaging by turning on the galvano mirrors and the photo multiplier.Observe the computer monitor to see the back scanner in signal of the gold nanoparticles. Maximize the back scanner in signal by adjusting the pinhole position and the height of the sample stage. After aligning the confocal microscope, characterize the scattering nonlinearity of the sample.To do this, first use a power meter to measure laser power after the objective lens. The reading should be less than 10 microwatts corresponding to an excitation intensity of less than 10 to the fourth watts per centimeter square. Then, acquire an image of the gold nanoparticles using the photo multiplier tube.Open the captured image in image analysis software to characterize the scattering intensity profile. Select a gold nanosphere among those in the image and draw a line across it. Then, follow the steps required in the software to retrieve the scattering profile.Fit the profile to a gaussian which allows a further check on the alignment of the imaging system. Now, begin to systematically scan through the excitation intensities. First, change the neutral density filter to increase the excitation intensity.Determine the new excitation intensity and record the back scattering image at the new intensity level. Use the image to extract a scattering profile of a nanosphere as before. Plot the scattering profile and identify its value at the center of the sphere as the scattering signal.Repeat these steps several times to build a plot of the scattering signal versus excitation intensity. In this figure, the blue points represent data. The red line is a polynomial fit.There is a linear relationship between the scattering signal and low values of the excitation energy. The drop below this linear relationship indicates that saturation has occurred. Next step is to perform spectroscopy on a single gold nanosphere.This schematic provides an overview of the setup. Use a super-continuum laser source with a wave length range of 450-1750 nanometers. Also, use a broadband 50/50 beamsplitter to ensure spectral coverage over the visible spectrum.Place a flipping mirror in front of the photo multiplier tube to direct the light toward the spectrometer. Which is equipped with a charge-coupled device. The super-continuum laser should be aligned using the same procedure as the 532 nanometer laser.At the output of the super-continuum laser, take some precautions to remove excess infrared power from the system. Place visible light reflecting mirrors in the optical path right after the laser output, before sending the beam to the microscope. In conjunction with the mirrors, use beam dumps to collect the infrared radiation that might damage the system.With the laser aligned, acquire and image of the gold nanospheres. View the image and identify a single nanosphere for study. Fix the focus of the incident broadband light on to the chosen nanosphere.Next, insure that the spectrometer is in the optical path. In this position, the flipping mirror directs the incident light to the photo multiplier tube. Reorient the mirror to direct the incident light to the spectrometer.Proceed to collect data on the scattering spectrum of the single gold nanoparticle. As in this example, the measured spectrum will be a mixture of nanosphere scattering and background due to reflections. After taking the spectrum, return the flipping mirror.Turn it to direct light to the photo multiplier tube. Take another image to confirm that the gold nanoparticle has not changed position. Next, shift the focus of the broadband light to a point where there is no particle.Change the mirror again to direct light to the spectrometer and proceed to make another spectrum measurement. The new spectrum is of the background. It will be subtracted from the spectrum of the gold nanosphere.Here is the clear back scattering spectrum produced by subtracting the background spectrum from the spectrum with the gold nanospheres. Saturated excitation microscopy of the sample requires a different setup. This is the schematic of the saturated excitation microscope.The laser source is a 532 nanometer laser. Use a 50/50 beam splitter to create two beams from the laser source. Pass each beam through a separate acoustal optic modulator.The different modulator frequencies generate a beat frequency that will serve as the modulation frequency of the saturated excitation signals. Combine the first order diffracted beams from the acoustal optic modulators with another 50/50 beam splitter. To monitor temporal modulation, use a glass slide to split a small portion of the laser light off to a photo detector.To check the system, connect the photo detector to an oscilloscope. Make sure no sample is in place. Assess the photo detector signal on the oscilloscope.The signal should be a sinusoid at the beat frequency if the modulation and beam overlapping are correct. For this experiment, the expected beat frequency is 10 kilohertz. Make efforts to achieve as perfect a sinusoid as possible with minimal nonlinearity.The next step, is to incorporate a lock-in amplifier in to the system. For saturated excitation microscopy, the lock-in amplifier has input from the photo detector and the photo multiplier tube. Disconnect the output of the photo detector from the oscilloscope and connect it to the reference input of the amplifier.Connect the output of the photo multiplier tube to the lock-in amplifier as the signal input. Send output from the amplifier to a data acquisiton card. At this point, mount the sample on the microscope stage.Use the same sample with 80 nanometer gold nanospheres in oil sealed between two glass plates. Return to the lock-in amplifier while taking measurements. Set the amplifier to export the absolute magnitude of the voltage signal.In this case, by selecting R"in the channel 1 display panel. Change the harmonic component setting at the reference channel to obtain the saturated excitation single amplitudes. These color images are formed using custom software to synchronize and combine the signals from the lock-in amplifier and the driving voltages of the galvano motors.The scanning electron microscope image is for comparison. The images demonstrate resolution enhancement with different harmonic components. The measured spectrum of an 80 nanometer gold nanosphere is in red.The solid curve calculated using Mie theory, shows excellent agreement. The localized surface plasmon resonance is at 580 nanometers. Scattering images, above, and line profiles, below have different features as a function of the excitation intensity.At low intensity, the point spread function is a standard gaussian profile. Higher intensities result in the function flattening and widening indicating saturation. At higher values, the central intensity is not the maximum, resulting in a donut-shaped point spread function.Eventually, the central intensity becomes the peak again during reverse saturation. The blue data points in this plot of the central intensity at different excitation intensities reveal both saturation and reverse saturation behavior. The data points are fit to a 5th order polynomial plotted in red.This data can be used to extract the harmonic frequency components. The harmonic components can also be found directly using the lock-in amplifier. This left plot is of experimental data.The right plot is the result of calculation using the 5th order polynomial fit. Both plots exhibit dips at specific intensities along the curves. For example, three dips in the second harmonic.Also in each plot, for each harmonic order, the slope increases after the first dip. Once mastered, this technique can be done in less than three hours if you perform it properly. While attempting this procedure, it is important to check the quality of the sinusoidal modulation, the linearity of the detection system, and the mechanical stability of the sample.After watching this video, you should have a good understanding of how to achieve resolution enhancement based on nonlinear scattering from plasmonic nanostructures. This technique provides an aspiring sample for researchers in the field of super-resolution microscopy to explore novel contrast in other fundamental light-matter interactions.

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12.8K vues.  National Taiwan University. The overall goal of this experiment is to demonstrate resolution enhancement with non-linear scattering from plasmonic nanostructures. In the field of optical microscopy, contrast and the resolution are the most important factors. Our technique expand to the super resolution field with the new scattering based on contrast.The main advantage of this technique is that scattering from nanoparticles is strong, highly nonlinear, and non-bleaching. Demonstrating the procedure will be Hsuan L...