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.
