We acknowledge the funding support from the Science, Technology, and Innovation Commission of Shenzhen Municipality (No. JCYJ20180306174930894), Zhongshan Municipal Bureau of Science and Technology (2020AG003), and Research Grant Council of Hong Kong (Project 26303018). We also acknowledge Prof. Chi-Ming Che and his funding support from &#34;Laboratory for Synthetic Chemistry and Chemical Biology&#34; under the Health@InnoHK Program launched by Innovation and Technology Commission, The Government of Hong Kong Special Administrative Region of the People&#39;s Republic of China.

1. Optical setup
<ol>
	<li>Direct a 532 nm laser beam (Raman excitation source) into the flex port of the optical tweezer microscope (see Table of Materials).</li>
	<li>Align the 532 nm laser beam into the stereo double-layer pathways of the optical tweezer microscope with a 750 nm long-pass dichroic mirror to combine with the original trapping laser beams to focus on the sample chamber.</li>
	<li>Collect the backscattered light from the sample chamber using a 750 nm long-pass dichroic mirror and redirect it into a spectrometer containing a liquid-nitrogen-cooled charge-coupled device (CCD) camera (see Table of Materials). Place a 532 nm notch filter in front of the entrance slit of the spectrometer before spectral acquisition. 
	&#8203;NOTE: Eye protection must be used when the laser is turned on, and the laser beam must be contained within a safe area.</li>
</ol>
2. Fabrication of AgNPs
<ol>
	<li>Heat 50 mL of 1 mM AgNO3 aqueous solution in a round-bottomed flask while boiling.</li>
	<li>Add 1.0 mL of 0.1 M trisodium citrate solution dropwise into the boiled AgNO3 aqueous solution.</li>
	<li>Keep the mixture boiling for 16 min under constant stirring.</li>
	<li>Cool the mixture to room temperature. Yellowish color is observed.</li>
	<li>Centrifuge the AgNP colloids at 2000 &#215; g for 5 min at room temperature and then remove the supernatant using a pipette.</li>
	<li>Resuspend the AgNP colloids with 1 mL of de-ionized water (resistivity of 18.2 M&#937; cm).</li>
	<li>Repeat steps 2.5 and 2.6 thrice to remove the residual reducing agent.</li>
	<li>Characterize the size distribution of the AgNPs using scanning electron microscopy (SEM) and dynamic light scattering (DLS)33 to confirm the uniformity of the AgNPs (Figure 2). The AgNP concentration was estimated as 0.1 nM by UV absorbance34. 
	NOTES: Due to the low concentration, the AgNP stock solution can be maintained without clustering for 2-3 weeks. No stabilizing agents are required. If a precipitate was observed in the AgNP stock solution, a new AgNP solution was prepared following the above protocol.</li>
</ol>
3. Interaction of the DSNB analyte molecule and AgNP
<ol>
	<li>Add 200 &#181;L of 2 mM DSNB (see Table of Materials) to 1 mL of AgNP colloid and incubate at room temperature for 3 h to coat a layer of DSNB on the surface of AgNP by the formation of the Ag-S bond between AgNP and DSNB35. A schematic representation of this interaction is shown in Figure 3.</li>
	<li>Centrifuge the AgNP at 2,000 &#215; g for 5 min at room temperature and remove the supernatant.</li>
	<li>Resuspend the AgNP-DSNB with 1 mL of de-ionized water.</li>
	<li>Repeat steps 3.2 and 3.3 thrice to remove excess DSNB.</li>
	<li>Record the UV-visible spectra of the AgNP colloid and AgNP-DSNB solution. 
	&#8203;NOTE: This spectra show an absorption peak shift from approximately 420 nm to 450 nm, indicating the successful coating of DSNB on the surface of AgNP (Figure 3).</li>
</ol>
4. Preparation of sample chamber and generation of AgNP assembly for SERS measurement
<ol>
	<li>Clean the glass slide and coverslip with water and ethanol.</li>
	<li>Attach the frame tape (0.25 mm thickness, see Table of Materials) to the glass slide to create a chamber (1.0 cm length &#215; 1.0 cm width &#215; 0.25 mm height).</li>
	<li>Add a few drops of the AgNP-DSNB solution (around 25 &#181;L) into the frame.</li>
	<li>Put the coverslip on the frame tape and seal it (Figure 4).</li>
	<li>Add liquid nitrogen to the container of the liquid nitrogen-cooled CCD camera until the temperature reaches -120 &#176;C.</li>
	<li>Block the Raman probe beam path using a magnetic laser safety screen (see Table of Materials), then turn on the 532 nm Raman excitation source laser.</li>
	<li>Fix the sample chamber with the AgNP-DSNB solution on the chamber holder. Add water to the water-immersed objective (60x magnification with a 1.2 numerical aperture A of 1.2) as shown in Figure 1. Then place the chamber holder immediately onto the microstage above the objective.</li>
	<li>Drop immersion oil on top of the coverslip and position the oil-immersed condenser to visualize particles on the microscope camera.</li>
	<li>Adjust the Z position of the objective by turning the knob of the microscope until the 532 nm Raman probe beam is focused on the bottom glass surface of the chamber, showing a white spot on the microscope camera (Figure 5).
	<ol>
		<li>Adjust the X- and Y-positions of the microstage to move the chamber to place the central region of the chamber at the white spot. Open the optical tweezer control software (see Table of Materials) and use the equipped joystick control to move the 1064 nm trapping laser (indicated by a red circle in the optical tweezer system) to overlap with the white spot (Figure 5).</li>
		<li>Next, tune the knob of the microscope to move the Z position of the objective up. 
		NOTE: The disappearance of the white spot in the microscope camera image indicates that the 532 nm Raman probe beam is focused inside the chamber.</li>
	</ol>
	</li>
	<li>Turn on the 1064 nm trapping laser to attract AgNPs in the sample chamber and create a plasmonic AgNP assembly. 
	NOTE: The gathering of AgNPs results in a dark spot in the sample chamber (Figure 6B).
	<ol>
		<li>Turn down the trapping laser beam to avoid overheating or bubble formation when required. 
		NOTE: Increase the trapping laser power and irradiation time if there is no apparent formation of the AgNP assembly.</li>
	</ol>
	</li>
	<li>Adjust the position of the sample microstage to place the dark spot of the plasmonic AgNP assembly under the focus of the 532 nm Raman probe beam for spectroscopic measurements.</li>
	<li>Place the neutral-density (ND) filters in front of the 532 nm Raman laser outlet to adjust the power to 10 mW. Input the acquisition time (10 s for the present study, Figure 6) in the setting panel in the spectrum software (see Table of Materials) and click on the Acquire button to start the spectral acquisition. 
	NOTE: This generates the SERS spectrum of the analyte molecules (DSNB in the representative result and Figure 6).</li>
</ol>

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The authors have nothing to disclose.

The present study reports an analytical platform that combines optical trapping and SERS detection for in situ molecular characterizations. A 532 nm Raman probe beam was combined with a 1064 nm trapping laser beam through stereo double-layer pathways to combine focus and collect for additional spectroscopic measurements in backscattering geometry. The trapping laser beam assembled AgNPs to form plasmonic hotspots, followed by excitation of the Raman probe laser beam to generate the SERS signal of the analyte molecules in solution. As a proof of concept, the detection of DSNB was demonstrated, which was coated on the surface of AgNPs. In the AgNP assembly region controlled by the trapping laser beam, an approximately 50-fold enhancement in the signal of DSNB compared to the surrounding dispersed AgNPs, was achieved. A similar high-signal amplification of analyte molecules in the solution-phase SERS measurements on the presented platform was reproducibly obtained.
The critical step affecting SERS signal amplification is forming an optical trapping-induced AgNP assembly. The SERS signal of the analyte molecules can be optimized by fine-tuning experimental parameters such as the trapping laser power, irradiation time, and AgNP concentration. As shown in Figure 8, using a higher trapping laser power can increase the efficiency of AgNP assembly formation. Reproducible AgNP assemblies were obtained by increasing the power of the trapping laser from 450 mW to 700 mW. However, a trapping laser power higher than 950 mW may induce overheating and bubble generation38. Thus, moderate trapping laser power is recommended to create a dynamic AgNP assembly. Analogously, a longer irradiation time is useful for promoting the formation of AgNP assemblies. Figure 8B shows that a clear spherical AgNP assembly was formed when the irradiation time increased from 5-20 s. However, the AgNP assembly was distorted after 60 s irradiation. In addition, the formation of the AgNP assembly was accelerated at a higher AgNP concentration, from 0.01 nM to 0.05 nM, while it was quickly overheated at 0.25 nM, as shown in Figure 8C. If there is no apparent AgNP assembly formation, increasing the trapping laser power and the irradiation time is recommended. Upon generation of a stable AgNP assembly, the trapping laser must be turned down to avoid potential thermal damage.
The SERS activity of the optical trapping-induced AgNP assembly was attributed to an increase in the local AgNP concentration in the trapping laser irradiation region, which is the dark spot in Figure 6B. In the fluidic AgNP solution, the optical trap can continuously attract AgNPs to accumulate and form plasmonic hotspots in a confined space in the interparticle junctions. This yields an enhanced electric field which enhances the SERS effect. It was further verified by the stronger SERS signal obtained at a higher AgNP concentration (1.00 nM) compared to the weaker SERS signal acquired at a lower AgNP concentration (0.05 nM) without the trapping laser, as shown in Figure 6E.
Furthermore, position control of the plasmonic AgNP assembly in solution, against Brownian motion, by optical trapping has significantly improved the efficiency and stability of SERS measurements. High-throughput sensing can be conducted when connected to the microfluidic system. Compared to the traditional salt-induced aggregation of nanoparticles to generate SERS-active substrates, our platform allows the dynamic formation of plasmonic AgNP assemblies, at the designed location and moment, with high flexibility26,28. Moreover, it works efficiently at nanomolar AgNP concentrations and enables the spatial-temporal manipulation of SERS-active hotspots for in situ spectroscopic measurements in solutions. This dynamic AgNP assembly gradually disassembled in a few minutes when the trapping laser was turned off. Without the trapping laser, the AgNP assembly almost disappeared in 20 min, as shown in Supplementary Figure 1. This can minimize the influence on the detection system and exhibits great potential for various bio-applications, especially the detection of biomolecules (DNA, RNA, and protein) under physiological and in vivo conditions. However, this dynamic AgNP assembly provides a smaller enhancement factor than salt-induced AgNP aggregates2, and hence, further modification and development are required.
In conclusion, the integration of optical trapping and SERS detection provides a convenient method to control plasmonic nanoparticles and achieve reproducible SERS signal enhancement to detect analyte molecules in solutions with high efficiency, stability, and biocompatibility.

Surface-enhanced Raman spectroscopy (SERS) is a sensitive analytical technique for directly detecting the chemical structure of target molecules at ultralow concentrations or even at the single-molecule level1,2,3,4. Laser irradiation induces localized surface plasmon resonance in metallic nanostructures, used as SERS substrates to amplify the Raman signals of target molecules. Salt-induced nanoparticle aggregates are the widely used SERS substrates, which spontaneously undergo Brownian motion in colloidal suspension liquids5,6. Further drying allows stable SERS measurements; however, impurity concentration may occur, which introduces background noise and causes irreversible damage to biological samples7. Hence, it is pertinent to develop salt-free nanoparticle aggregations, control their movement in solution, and improve biocompatibility while maintaining measurement efficiency.
Optical trapping has been adopted to control various metallic substrates and facilitate SERS detections8,9,10,11,12,13,14. An optical trap is generated by tightly focusing a laser beam to generate an optical force field, which attracts small objects to the highest-intensity region around the focus15,16. Recently, optical traps have been used to develop reproducible and sensitive plasmonic sensing platforms for various applications, displaying their unique advantages in locating and controlling the position of SERS-active metallic nanostructures in solutions17,18,19,20,21,22,23,24. The present protocol introduces an approach to combine optical tweezers and Raman spectro-microscopy to dynamically assemble silver nanoparticles (AgNPs) and stabilize them against Brownian motion in solution for efficient SERS measurements. In the AgNP assembly region, the signal of the 3,3&#39;-dithiobis[6-nitrobenzoic acid] bis(succinimide) ester (DSNB), analyte molecules coated on the surface of AgNPs, can be enhanced by approximately 50 folds. This approach is suitable for analyzing sensitive biomolecules incompatible with chemical capping agents25,26,27. Moreover, it provides spatial and temporal control to generate the SERS-active AgNP assembly. This enables in situ detection in aqueous environments, which could lower the usage of AgNPs and minimize perturbation for in vivo analysis28,29,30. In addition, the optical trapping-induced AgNP assembly is stable, reproducible, and reversible31,32. Hence, it is a promising platform for detecting analyte molecules in solutions and under physiological conditions where salt-induced aggregation is not applicable.
In the present study, a 1064 nm trapping laser, force detection module, and brightfield illumination source are integrated into the optical tweezer microscopy system for optical manipulation and visualization of particles. A 532 nm Raman probe laser was also incorporated into the microscope and aligned with the trapping laser in the sample chamber. For spectral acquisition, backscattered light was collected and redirected into a spectrometer (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1064 nm trapping laser</td>
			<td>IPG Photonics, United States</td>
			<td>1064 nm CW Yb fiber laser, 10W</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3&#39;-Dithiobis[6-nitrobenzoic acid] bis(succinimide) ester&#160;</td>
			<td>Biosynth Carbosynth</td>
			<td>FD15467</td>
			<td></td>
		</tr>
		<tr>
			<td>532 nm Raman excitation source</td>
			<td>CNI, China</td>
			<td>MLL-III-532</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluelake software</td>
			<td>LUMICKS, Netherlands</td>
			<td>version 1.6.12</td>
			<td>optical tweezer control software</td>
			<td></td>
		</tr>
		<tr>
			<td>Frame tape</td>
			<td>Thermo Fisher Scientific, Inc</td>
			<td>AB-0576</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion oil</td>
			<td>Cargille Laboratories, Inc</td>
			<td>16482</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen-cooled charge-coupled device (CCD) camera</td>
			<td>Teledyn Princeton Instrument, United States</td>
			<td>400B eXcelon</td>
			<td></td>
		</tr>
		<tr>
			<td>Long-pass dichroic mirror</td>
			<td>AHF, Germany</td>
			<td>F48-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic laser safety screen</td>
			<td>ThorLabs</td>
			<td>TPSM2</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical tweezer microscope</td>
			<td>LUMICKS, Netherlands</td>
			<td>m-trap</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma-Aldrich China, Inc.</td>
			<td>S8157</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Teledyn Princeton Instrument, United States</td>
			<td>IsoPlane SCT-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate</td>
			<td>Sigma-Aldrich China, Inc.</td>
			<td>S4641</td>
			<td></td>
		</tr>
		<tr>
			<td>WinSpec software</td>
			<td>Teledyn Princeton Instrument, United States</td>
			<td>version 2.6.24.0</td>
			<td>spectrum software</td>
		</tr>
	</tbody>
</table>

optical trapping of plasmonic nanoparticles for in situ surface-enhanced raman spectroscopy characterizations

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric field of metallic nanostructures. Salt-induced silver nanoparticle aggregation is the most popular method for generating SERS-active substrates; however, it is limited by poor reproducibility, stability, and biocompatibility. The present protocol integrates optical manipulation and SERS detection to develop an efficient analytical platform to address this. A 1064 nm trapping laser and a 532 nm Raman probe laser are combined in a microscope to assemble silver nanoparticles, which generate plasmonic hotspots for in situ SERS measurements in aqueous environments. Without aggregating agents, this dynamic plasmonic silver nanoparticle assembly enables an approximately 50-fold enhancement of the analyte molecule signal. Moreover, it provides spatial and temporal control to form the SERS-active assembly in as low as 0.05 nM analyte-coated silver nanoparticle solution, which minimizes the potential perturbation for in vivo analysis. Hence, this optical trapping-integrated SERS platform holds great potential for efficient, reproducible, and stable molecular analyses in liquids, especially in aqueous physiological environments.

As proof of concept, DSNB was chosen as the analyte molecule and coated onto the surface of AgNPs. The typical SERS spectra of DSNB enhanced by the plasmonic AgNP assembly and dispersed AgNP are shown in Figure 6. Without the trapping laser, the dispersed AgNPs in the sample chamber generated a black spectrum (Figure 6A) upon excitation by the Raman probe laser. A weak and broad SERS signal was observed at approximately 1380-1450 cm-1, the characteristic peak of DSNB from its symmetric NO2 stretch, which is consistent with literature reports35,36. Since the dispersed AgNPs were under Brownian motion, the interparticle junctions were large and unstable, as illustrated in Figure 6C. Thus, the SERS signal amplification of DSNB was low for the dispersed AgNPs.
AgNPs are gathered to form a plasmonic AgNP assembly when the trapping laser is on. Increasing the power and extending the irradiation time of the trapping laser could attract more AgNPs and generate a dark spot, as shown in Figure 6B. Here, we applied a trapping laser power of 700 mM and a 20 s irradiation time to create a plasmonic AgNP assembly in a 0.05 nM DSNB-coated AgNP solution at a designated location and moment. The SERS spectrum of DSNB was obtained in the region of the plasmonic AgNP assembly (Figure 6A, red). The strong Raman band at 930 cm-1 is assigned to the nitro scissoring vibration, and the large bands at 1078 cm-1, 1152 cm-1, and 1191 cm-1 likely correspond to the succinimidyl N-C-O stretch overlapping with the aromatic ring modes of DSNB35,37. The feature bands at 1385 cm-1 and 1444 cm-1 arise from the symmetric nitro stretch of DSNB and are significantly enhanced and slightly shifted due to the reaction with the surface of AgNP35,37. Based on the previously reported SERS fingerprints of DSNB35,36,37, the band at 1579 cm-1 was assigned to the aromatic ring mode of DSNB. The overall intensities of DSNB in the plasmonic AgNP assembly were higher than those of the dispersed AgNP. Considering the intensity of the characteristic peak at 1444 cm-1, the plasmonic AgNP assembly can provide approximately a 50-fold enhancement of the SERS signal of DSNB compared to that of the dispersed AgNP. As shown in Figure 7, SERS spectra of DSNB were recorded repeatedly (20 times) for the AgNP assembly in the experiment, demonstrating identical vibrational features. The intensities of the characteristic peaks of DSNB at 1152 cm&#8722;1, 1444 cm&#8722;1, and 1579 cm&#8722;1 across these 20 SERS spectra were plotted as histograms with relative standard deviations (RSD) of 6.88%, 6.59%, and 5.48%, respectively. This further verified the reproducibility and stability. Hence, this approach is reliable for manipulating plasmonic nanoparticles and SERS detection of analyte molecules in solution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63862/63862fig01.jpg" /> 
Figure 1: Schematic representation of the optical tweezer-coupled Raman spectroscopic platform. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63862/63862fig02.jpg" /> 
Figure 2: Preparation of AgNP for SERS measurement. (A) SEM image of AgNP. (B) Size distribution of AgNP by DLS. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63862/63862fig03.jpg" /> 
Figure 3: Interaction of AgNP and DSNB. (A) Schematic of the coating of DSNB on the surface of AgNP. (B) UV-visible spectra of AgNP and AgNP-DSNB. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63862/63862fig04.jpg" /> 
Figure 4: Schematic of sample chamber preparation. (A) Sample chamber preparation process. (B) Prepared sample chamber. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63862/63862fig05.jpg" /> 
Figure 5: Position overlapping of 532 nm Raman laser and 1064 nm trapping laser. (A) Position of 532 nm Raman laser indicated by white spot. (B) Position of 1064 nm trapping laser indicated by red circle. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63862/63862fig06.jpg" /> 
Figure 6: Typical SERS spectra of the analyte molecules enhanced by the plasmonic AgNP assembly. (A) SERS spectra of DSNB at the plasmonic AgNP assembly (red) and the dispersed AgNP (black). (B) The plasmonic AgNP assembly when the trapping laser is on shows a dark spot under microscopic visualization. (C) The dispersed AgNP when the trapping laser is off. (D) Illustration of the mechanism of AgNP assembly formation. (E) Concentration-dependent SERS intensity in the absence of the trapping laser. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63862/63862fig07.jpg" /> 
Figure 7: Reproducibility of SERS signal of DSNB. (A) 20 SERS spectra of DSNB at the plasmonic AgNP assembly recorded repeatly in the experiment. (B) Histograms of the intensities of the characteristic DSNB peaks at 1152 cm-1 (RSD = 6.88%), 1444 cm-1 (RSD = 6.59%), and 1579 cm-1 (RSD = 5.48%). <a href="https://www.jove.com/files/ftp_upload/63862/63862fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63862/63862fig08.jpg" /> 
Figure 8: AgNP assembly generated under different experimental parameters. (A) Different trapping laser power; irradiation time 20 s and AgNP concentration 0.05 nM. (B) Different irradiation time; trapping laser power 700 mW and AgNP concentration 0.05 nM. (C) Different AgNP concentration; irradiation time 20 s and trapping laser power 700 mW. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: The microscope camera images of AgNP assembly in time series when the trapping laser was turned off. <a href="https://www.jove.com/files/ftp_upload/63862/Figure S1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations at JoVE.com

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric ...

optical-trapping-plasmonic-nanoparticles-for-situ-surface-enhanced

Research

JoVE Journal

Bioengineering

2.5K Views.  HKUST-Shenzhen Research Institute. The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Video: Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.