The author thanks Prof. Y. Ozaki (Kwansei Gakuin University) and Dr. T. Itoh (National Institute of Advanced Industrial Science and Technology) for their fruitful discussion of this work. This work was supported by KAKENHI (Grant-in-Aid for Scientific Research C) from the Ministry of Education, Culture, Sports, Science, and Technology (No. 16K05671).

1. Sample Preparation
<ol>
	<li>Preparation of silver colloidal nanoparticles20
	<ol>
		<li>To fabricate silver colloidal nanoparticles, dissolve 0.030 g of silver nitrate and 0.030 g of trisodium citrate dihydrate in 150 mL of water in a 200-mL round bottom flask.</li>
		<li>Combine the flask with a reflux (Dimroth) condenser.</li>
		<li>Stir the solution in the flask with a magnetic stirrer and stir bar. Then, heat the stirring solution in the flask in an oil bath at 150 &#176...

<ol>
	<li>Qian, X. M., Nie, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+and+single-nanoparticle+SERS:+from+fundamental+mechanisms+to+biomedical+applications.">Single-molecule and single-nanoparticle SERS: from fundamental mechanisms to biomedical applications.</a> Chem. Soc. Rev. 37, 912-920 (2008).</li><li>Pieczonka, N. P. W., Aroca, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+analysis+by+surfaced-enhanced+Raman+scattering.">Single molecule analysis by surfaced-enhanced Raman scattering.</a> Chem. Soc. Rev. 37, 946-954 (2008).</li><li>Kneipp, J., Kneipp, H., Kneipp, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SERS+-a+single-molecule+and+nanoscale+tool+for+bioanalytics.">SERS -a single-molecule and nanoscale tool for bioanalytics.</a> Chem. Soc. Rev. 37, 1052-1060 (2008).</li><li>Kitahama, Y., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blinking+SERS+by+a+power+law+with+an+exponential+function.">Analysis of blinking SERS by a power law with an exponential function.</a> Frontiers of Surface-Enhanced Raman Scattering: Single-Nanoparticles and Single Cells. , (2014).</li><li>Kitahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncated+Power+Law+Analysis+of+Blinking+SERS.">Truncated Power Law Analysis of Blinking SERS.</a> Frontiers of Plasmon Enhanced Spectroscopy Volume 1 (ACS Symposium series Vol. 1245). , (2016).</li><li>Bizzarri, A. R., Cannistraro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L&#233;vy+Statistics+of+Vibrational+Mode+Fluctuations+of+Single+Molecules+from+Surface-Enhanced+Raman+Scattering.">L&#233;vy Statistics of Vibrational Mode Fluctuations of Single Molecules from Surface-Enhanced Raman Scattering.</a> Phys. Rev. Lett. 94, 068303 (2005).</li><li>Kitahama, Y., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power-law+analysis+of+surface-plasmon-enhanced+electromagnetic+field+dependence+of+blinking+SERS+of+thiacyanine+or+thiacarbocyanine+adsorbed+on+single+silver+nanoaggregates.">Power-law analysis of surface-plasmon-enhanced electromagnetic field dependence of blinking SERS of thiacyanine or thiacarbocyanine adsorbed on single silver nanoaggregates.</a> Phys. Chem. Chem. Phys. 13, 7439-7448 (2011).</li><li>Kitahama, Y., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+excitation+laser+intensity+dependence+of+blinking+SERRS+of+thiacarbocyanine+adsorbed+on+single+silver+nanoaggregates+by+using+a+power+law+with+an+exponential+function.">Analysis of excitation laser intensity dependence of blinking SERRS of thiacarbocyanine adsorbed on single silver nanoaggregates by using a power law with an exponential function.</a> Chem. Commun. 47, 3888-3890 (2011).</li><li>Kitahama, Y., Enogaki, A., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncated+power+law+analysis+of+blinking+SERS+of+thiacyanine+molecules+adsorbed+on+single+silver+nanoaggregates+by+excitation+at+various+wavelengths.">Truncated power law analysis of blinking SERS of thiacyanine molecules adsorbed on single silver nanoaggregates by excitation at various wavelengths.</a> J. Phys. Chem. C. 117, 9397-9403 (2013).</li><li>Kitahama, Y., Araki, D., Yamamoto, Y. S., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+behaviour+of+molecules+in+dark+SERS+state+on+colloidal+Ag+nanoparticles+estimated+by+truncated+power+law+analysis+of+blinking+SERS.">Different behaviour of molecules in dark SERS state on colloidal Ag nanoparticles estimated by truncated power law analysis of blinking SERS.</a> Phys. Chem. Chem. Phys. 17, 21204-21210 (2015).</li><li>Kitahama, Y., Nagahiro, T., Tanaka, Y., Itoh, T., Ozaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blinking+from+multicoloured+SERS-active+Ag+colloidal+nanoaggregates+with+poly-L-lysine+via+truncated+power+law.">Analysis of blinking from multicoloured SERS-active Ag colloidal nanoaggregates with poly-L-lysine via truncated power law.</a> J. Raman. Spectrosc. 48, 570-577 (2017).</li><li>Habuchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Surface+Enhanced+Resonance+Raman+Spectroscopy+of+the+Enhanced+Green+Fluorescent+Protein.">Single-Molecule Surface Enhanced Resonance Raman Spectroscopy of the Enhanced Green Fluorescent Protein.</a> J. Am. Chem. Soc. 125, 8446-8447 (2003).</li><li>Weiss, A., Haran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-Dependent+Single-Molecule+Raman+Scattering+as+a+Probe+of+Surface+Dynamics.">Time-Dependent Single-Molecule Raman Scattering as a Probe of Surface Dynamics.</a> J. Phys. Chem. B. 105, 12348-12354 (2001).</li><li>Emory, S. R., Jensen, R. A., Wenda, T., Han, M., Nie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-examining+the+origins+of+spectral+blinking+in+single-molecule+and+single-nanoparticle+SERS.">Re-examining the origins of spectral blinking in single-molecule and single-nanoparticle SERS.</a> Faraday Discuss. 132, 249-259 (2006).</li><li>Itoh, T., Iga, M., Tamaru, H., Yoshida, K., Biju, V., Ishikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+blinking+in+surface+enhanced+resonance+Raman+scattering+and+fluorescence+by+electromagnetic+mechanism.">Quantitative evaluation of blinking in surface enhanced resonance Raman scattering and fluorescence by electromagnetic mechanism.</a> J. Chem. Phys. 136, 024703 (2012).</li><li>Willets, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+of+SERS+hot+spots.">Super-resolution imaging of SERS hot spots.</a> Chem. Soc. Rev. 43, 3854-3864 (2014).</li><li>Dieringer, J. A., Lettan, R. B., Scheidt, K. A., Van Duyne, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Frequency+Domain+Existence+Proof+of+Single-Molecule+Surface-Enhanced+Raman+Spectroscopy.">A Frequency Domain Existence Proof of Single-Molecule Surface-Enhanced Raman Spectroscopy.</a> J. Am. Chem. Soc. 129, 16249-16256 (2007).</li><li>Cichos, F., von Borczyskowski, C., Orrit, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power-law+intermittency+of+single+emitters.">Power-law intermittency of single emitters.</a> Curr. Opin. Colloid Interface Sci. 12, 272-284 (2007).</li><li>Tang, J., Marcus, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fluorescence+blinking+in+semiconductor+nanocrystal+quantum+dots.">Mechanisms of fluorescence blinking in semiconductor nanocrystal quantum dots.</a> J. Chem. Phys. 123, 054704 (2005).</li><li>Lee, P. C., Meisel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+surface-enhanced+Raman+of+dyes+on+silver+and+gold+sols.">Adsorption and surface-enhanced Raman of dyes on silver and gold sols.</a> J. Phys. Chem. 86, 3391-3395 (1982).</li><li>Krichevsky, O., Bonnet, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+correlation+spectroscopy:+the+technique+and+its+applications.">Fluorescence correlation spectroscopy: the technique and its applications.</a> Rep. Prog. Phys. 65, 251-297 (2002).</li><li>Hess, S. T., Huang, S., Heikal, A. A., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+Chemical+Applications+of+Fluorescence+Correlation+Spectroscopy:+A+Review.">Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A Review.</a> Biochemistry. 41, 697-705 (2002).</li></ol>

The author has nothing to disclose.

From the silver nanoaggregate junction, SERS is emitted. Thus, we need to prepare nanoaggregates rather than colloidal nanoparticles, which are covered with citrate anions. Silver aggregates are formed from the salting out effect created by the addition of poly-L-lysine, which has -NH3+ and is the origin of the SERS, or Na+ cations from NaCl, as shown in Figure S2 of the supplementary material. Moreover, to illuminate the many spots in the wide area, the unfocused laser b...

Surface-enhanced Raman scattering (SERS) is highly sensitive Raman spectroscopy from a noble metal surface. Since the Raman spectrum provides detailed information about molecular structure based on the sharp peak positions, through the vibrational modes of functional groups in the molecules, the information of a single molecule on a metal surface can be investigated using SERS1,2,3. From a silver nanoaggregate with an adsorbate at the single-molecule level, a blinking signal is observed1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16, and the spectrum fluctuates1,2,3,4,5,6,7,8,9,10,11,12,13,14. Blinking can be induced by a single molecule that randomly moves in and out of an enhanced electromagnetic (EM) field at a nanometer-sized silver nanoaggregate junction. Therefore, blinking is considered simple evidence for single-molecule detection, compared with a technique using a Poisson distribution of SERS intensities and a bi-analyte2,3,17. However, the detailed mechanisms of the blinking and fluctuating spectrum, which may strongly depend on molecular behavior on the Ag surface, are still controversial.
In previous studies, blinking SERS has been analyzed using the autocorrelation function, which can calculate the diffusion coefficient and concentration of molecules moving in and out of an enhanced EM field12,13,14. Moreover, a normalized standard deviation score, which represents instability in the total intensity, has been derived from the time profile of the signal15. However, these analytic approaches may be based on the behavior of a few molecules. In contrast, in a super-resolution imaging of blinking SERS, single-molecule behavior in an enhanced EM field can be identified16. However, these techniques can obtain such parameters only in an enhanced EM field. The random behavior of a single molecule within a wide range (for instance, in blinking SERS) can be represented as a power law rather than an average4,5,6,7,8,9,10,11, similar to blinking fluorescence from a single semiconductor quantum dot (QD)18,19. By using a power law analysis4,5,6,7,8,9,10,11, molecular behavior can be estimated in both the bright state (in the enhanced EM field) and dark state10; that is, the behavior of the molecule over the entire silver surface can be estimated.
For this technique, silver colloidal nanoaggregates are used4,5,6,7,8,9,10,11. These nanoaggregates show various localized surface plasmon resonance (LSPR) bands that strongly affect enhanced electromagnetic fields when they are excited at certain wavelengths. Thus, SERS-active silver nanoparticles exist in colloidal suspension, and some data can immediately be obtained. In the case of simple nanostructures, which have specific sizes, shapes, and arrangements, the LSPR dependence of SERS blinking can conceal other dependences7; namely, if the good or bad nanostructure to LSPR is used, the parameters will be constant, and the other dependences will therefore be hidden. Power law analysis has been used to discover various dependences of the blinking SERS from silver colloidal nanoaggregates4,5,6,7,8,9,10,11.

<table border="0" cellpadding="0" cellspacing="0" style="width:639px;" width="638">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Silver nitrate, 99.8%</td>
			<td style="width:83px;">Wako</td>
			<td style="width:119px;">194-00832</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Trisodium citrate dihydrate, 99. %</td>
			<td style="width:83px;">Wako</td>
			<td style="width:119px;">191-01785</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Poly-L-lysine aqueous solution, 0.1%</td>
			<td style="width:83px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="116">
			<td height="116" style="height:116px;width:227px;">3,3&#39;-disulfopropylthiacyanine triethylamine</td>
			<td style="width:83px;">Hayashibara Biochemical Laboratories</td>
			<td style="width:119px;">NK-2703</td>
			<td>a kind of thiacyanine dyes</td>
		</tr>
		<tr height="116">
			<td height="116" style="height:116px;width:227px;">3,3&#39;-diethyl-5,5&#39;-dichloro-9-methylthiacarbocyanine iodine salt</td>
			<td style="width:83px;">Hayashibara Biochemical Laboratories</td>
			<td style="width:119px;">SMP-9</td>
			<td>a kind of thiacarobocyanine dyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Sodium chloride, 99.5%</td>
			<td style="width:83px;">Wako</td>
			<td style="width:119px;">191-01665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Dimroth condenser</td>
			<td style="width:83px;">Iwaki</td>
			<td style="width:119px;">61-9722-22</td>
			<td>perchased from AS ONE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Magnetic stirrer</td>
			<td style="width:83px;">Corning</td>
			<td style="width:119px;">DC-420D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Oil bath</td>
			<td style="width:83px;">Advantech</td>
			<td style="width:119px;">OS-220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Glass plate</td>
			<td style="width:83px;">Matsunami</td>
			<td style="width:119px;">S-1112</td>
			<td>Microscope slide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Blower</td>
			<td style="width:83px;">Hozan</td>
			<td style="width:119px;">Z-288</td>
			<td>Air duster</td>
		</tr>
		<tr height="116">
			<td height="116" style="height:116px;width:227px;">Liquid blocker pen</td>
			<td style="width:83px;">Daido Sangyo</td>
			<td style="width:119px;"></td>
			<td style="width:211px;">LIQUID BLOCKER (Super Pap Pen). Ready-to-use hydrophobic barrier pen designed for immunohistochemistry applications</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Inverted microscope</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:119px;">IX-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Objective lens</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:119px;">LCPlanFl 60&#215;</td>
			<td>NA 0.7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Dark field condenser</td>
			<td style="width:83px;">Olympus</td>
			<td style="width:119px;">U-DCD</td>
			<td style="width:211px;">NA 0.8&#8211;0.92</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Cooled digital CCD camera</td>
			<td style="width:83px;">Hamamatsu</td>
			<td style="width:119px;">ORCA-AG</td>
			<td style="width:211px;">controlled by software Aqua Cosmos</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:227px;">Software for the cooled digital CCD camera</td>
			<td style="width:83px;">Hamamatsu</td>
			<td style="width:119px;">AquaCosmos</td>
			<td style="width:211px;">used for also derivation of the time-profiles from the blinking spots in the video&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Color CCD camera</td>
			<td style="width:83px;">ELMO</td>
			<td style="width:119px;">TNC-C920</td>
			<td>not used for analysis</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:227px;">DPSS laser</td>
			<td style="width:83px;">RGB laser system</td>
			<td style="width:119px;">NovaPro532-75</td>
			<td style="width:211px;">&#955; = 532 nm; 
			60 mW (corresponds to a power density of 600 W/cm2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Interference filter</td>
			<td style="width:83px;">Semrock</td>
			<td style="width:119px;">LL01-532-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Long pass filter</td>
			<td style="width:83px;">Semrock</td>
			<td style="width:119px;">BLP01-532R-25</td>
			<td></td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:227px;">Software for the distinguishment and counting of the bright/dark events</td>
			<td style="width:83px;">home-maid</td>
			<td style="width:119px;"></td>
			<td>programmed by C++</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:227px;">Software for the fitting by a power law</td>
			<td style="width:83px;">LightStone</td>
			<td style="width:119px;">Origin6.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

observation and analysis of blinking surface-enhanced raman scattering

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on how to prepare the SERS-active silver nanoaggregate, record a video of certain blinking spots in the microscopic image, and analyze the blinking statistics. In this analysis, a power law reproduces the probability distributions for bright events relative to their duration. The probability distributions for dark events are fitted by a power law with an exponential function. The parameters of the power law represent molecular behavior in both bright and dark states. The random walk model and the speed of the molecule across the entire silver surface can be estimated. It is difficult to estimate even when using averages, autocorrelation functions, and super-resolution SERS imaging. In the future, power law analyses should be combined with spectral imaging, because the origins of blinking cannot be confirmed by this analysis method alone.

From the silver nanoaggregates with poly-L-lysine prepared by protocol 1.2, multicolored blinking spots from SERS and surface-enhanced fluorescence are observed, as shown in Figure 111. In contrast, monotonous colored blinking spots from SERS were observed for the silver nanoaggregates with the dye molecules prepared by protocol 1.37,8,9,...

Watch this Scientific Journal Video about Observation and Analysis of Blinking Surface-enhanced Raman Scattering at JoVE.com

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

This protocol describes the analysis of blinking surface-enhanced Raman scattering due to the random walk of a single molecule on a silver surface using power laws.

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...

The overall goal of this analysis of Blinking Surface-enhanced Raman Scattering is to investigate the behavior of single molecules on metal nano-particle surfaces. This method can help answer key questions in interface and coral science about working behavior of solid liquid interfaces The main advantage of this technique is that many data can be gathered because approximately a dozen blinking spots can be simultaneously observed in one video. To begin this procedure, wash two glass microscope slides with liquid detergent and tap water.Rinse the slides with distilled water and allow them to air dry. Next, apply an aqueous solution of 0.1 percent poly-l lysine to one dry slide. Allow the solution to sit on the slide for two to three seconds.Then, use a compressed air blower to remove the excess solution and dry the slide. Next, apply a suspension of colloidal silver nanoparticles to the slide. Allow the suspension to sit for two to three seconds.Dry the slide with compressed air. Then, use a hydrophobic barrier pen to completely enclose a 26 millimeter by 26 millimeter area on the slide. Allow the barrier to dry.Drop distilled water onto the slide to fill the enclosed area. Cover the slide with the second clean dry glass slide to prevent water evaporation. Place the slide assembly on the sample stage of the inverted microscope.Illuminate the slide with white light through a dark field condenser. Focus on spots of various colors by changing from a 2x objective lens to a 60x objective lens. Next, use a diode pumped solid state continuous wave 532 nanometer laser, a band pass interference filter, and a lens to illuminate the sample with an attenuated beam at a 30 degree angle relative to the sample surface.Move the laser illumination to the center of the view, and slightly adjust the z direction of the sample stage, to refine the focus on the spots. Observe the aggregates as monotone spots against a uniform background. Next, turn off the laser, and insert a long pass-edge filter after the objective lens.Illuminate the sample with the laser at a 30 degree angle relative to the sample surface, through the interference filter and the external lens. Move the stage in the x and y directions to find the blinking spots. Then, use a deep cool digital CCD camera with a frame time of 61 to 120 milliseconds to acquire a 20 minute long video of the spots.To begin the analysis, open the acquired video in the CCD camera software. Click and drag to select areas with and without blinking spots. Perform a temporal analysis to derive a signal intensity time profile for the spots and dark areas.Acquire profiles for all blinking spots and dark areas of interest in this way. Then, generate probability distributions for bright and dark events relative to their durations. Multicolored blinking spots were observed from SERS of silver nano aggregates, prepared with poly-l lysine coated slides.The blinking signal is attributed to the random walk single molecules at silver nano aggregate junctions. The signals from the single nano aggregate showed varying intensities over time, unlike the blinking fluorescence of a quantum dot. The threshold for bright events was set to three standard deviations above the baseline intensity, and the bright and dark event durations were determined for each spot.The probability distribution of bright events was plotted against event duration as a line and a log log graph, whereas the probability distribution of dark events was plotted against duration as a curve. As the dark event durations were generally longer than the bright even durations, the power law exponents for bright events tended to be smaller than those for dark events. Shorter truncations times for dark events indicated a quicker molecular random walk, a higher energy barrier from a non-emissive state to an emissive state, or a combination of both.After watching this video, you should have a good understanding of how to observe and analyze blinking SERS phenomena.

observation-and-analysis-of-blinking-surface-enhanced-raman-scattering

Research

Education

JoVE Journal

JoVE Core

Statistics

Chemistry

Unit 1

Summarizing and Visualizing Data

SCIENTISTS IN ACTION

7.2K 조회수.  Kwansei Gakuin University. The overall goal of this analysis of Blinking Surface-enhanced Raman Scattering is to investigate the behavior of single molecules on metal nano-particle surfaces. This method can help answer key questions in interface and coral science about working behavior of solid liquid interfaces The main advantage of this technique is that many data can be gathered because approximately a dozen blinking spots can be simultaneously observed in one video. To begin this procedure, wash two glass microscop...