Name: observation and analysis of blinking surface-enhanced raman scattering
Uploaded: 2018-01-11T14:00:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Observation and Analysis of Blinking Surface-enhanced Raman Scattering at JoVE.com

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;C for 60 min. 
		NOTE: The solution will turn yellow, then milky grey.</li>
		<li>Cool the suspension at room temperature, and keep the suspension in the flask covered with aluminum foil in a refrigerator. 
		NOTE: The protocol can be paused at this point. Use the colloidal nanoparticles, after storing in a refrigerator, within one month.</li>
	</ol>
	</li>
	<li>Preparation of sample for multi-colored blinking emission11
	<ol>
		<li>To ready a microscope slide, wash a glass plate with soap by hand and rinse it with water.</li>
		<li>Add 0.1% poly-L-lysine aqueous solution to the glass plate, and remove the solution with a blower.</li>
		<li>Add the silver colloidal suspension to the glass plate, and remove the suspension with a blower.</li>
		<li>Enclose a drop area on the glass plate with a liquid blocker pen.</li>
		<li>Drop distilled water on the glass plate, and cover it with another glass plate to create a microscope slide and prevent water from evaporating.</li>
	</ol>
	</li>
	<li>Preparation of sample for monotonous colored blinking SERS7,8,9,10
	<ol>
		<li>To ready a microscope slide, wash a glass plate with soap by hand and rinse it with water.</li>
		<li>Mix the silver colloidal suspension with thiacyanine or thiacarbocyanine dye (25 or 4 &#181;M, respectively) and a NaCl (10 mM) aqueous solution at a volume ratio of 2:1:1.</li>
		<li>Drop the sample suspension onto the glass plate, and remove the suspension with a blower.</li>
		<li>Enclose a drop area on the glass plate with a liquid blocker pen.</li>
		<li>Drop an aqueous solution of NaCl (1 M) on the glass plate to immobilize the silver nanoparticles, and cover it with another glass plate to create a microscope slide plate and prevent the solution from evaporating.</li>
	</ol>
	</li>
</ol>
2. Observation of Blinking Silver Nanoparticles
<ol>
	<li>Illumination of sample
	<ol>
		<li>Place the sample glass plate prepared using protocol 1.2 or 1.3 on the stage of an inverted microscope.</li>
		<li>Illuminate the sample glass plate using white light through a dark field condenser, and focus on various colored spots (blue, green, yellow, and red) on the glass plate using an objective lens (60X).</li>
		<li>Illuminate the sample glass plate using an attenuated beam, delivered at an angle of 30&#176; relative to the sample surface, from a diode pumped solid state (DPSS) continuous-wave (cw) laser through an interference filter.</li>
		<li>To use laser illumination to observe the silver nanoaggregates as monotonous colored spots in a same-colored surrounding, move the laser illumination area to the center of the view, and focus on the spots on the glass plate by adjusting the stage in the z-direction.</li>
	</ol>
	</li>
	<li>Observation of blinking
	<ol>
		<li>Insert a long-pass filter after the objective lens, and illuminate the sample glass plate using a DPSS cw-laser beam delivered at an angle of 30&#176; relative to the sample surface through an interference filter.</li>
		<li>Find the blinking spots as shown in Figure 1 (see also Figure S1 in the supplementary material) by moving the stage in the x- and y-directions.</li>
		<li>Record video of the blinking spots with the inverted microscope, coupled to a cooled digital charge-coupled device (CCD) camera, which has a time resolution of 61 - 120 ms, for 20 min.</li>
	</ol>
	</li>
</ol>
3. Analysis of Blinking SERS
<ol>
	<li>Derivation of time profile from video
	<ol>
		<li>In the software that controls the CCD camera, open the video file.</li>
		<li>To select the blinking spots and dark area, drag areas that separately cover regions with and without spots in the video image.</li>
		<li>To derive signal intensity time profiles from the blinking spots and dark areas in the video, select Temporal Analysis in Analysis, and click the Calculate in the Temporal Analysis window.</li>
		<li>Save the data as a text file.</li>
	</ol>
	</li>
	<li>Analysis of the time profile
	<ol>
		<li>Flatten a baseline of the time profile by subtracting the time profile from the dark area and/or fitting with a polynomial function, as shown in Figures 2A and 2B.</li>
		<li>Evaluate an averaged baseline intensity that consists of approximately 2000 points, Ibase, and a standard deviation of baseline intensities, &#963;,&#160;as shown in Figures 2C and 2D.</li>
		<li>Distinguish bright events from dark events using larger intensities than a threshold of Ibase + 3&#963;,&#160;and record the duration of each event. In Figure 3, for example, record the event from 0 to 3.5476 s as the dark event (with a duration of t = 3.5476 s), and record the event from 3.5476 to 4.0981 s as the bright event (with a duration of t = 0.5505 s). Repeat procedure as shown in Table 1.</li>
		<li>Count the number of bright and dark events for each duration, as expressed in the first and second lines of Table 2.</li>
		<li>Sum the number of events for each duration, except for events shorter than duration t. As expressed in the second and third lines of Table 2, for example, sum the number of events for each duration (except for the events for t = 0.0612 s) as 41 + 18 + 9 + &#8230;; the result equals the summation for t = 0.1223 s, i.e. 103.</li>
		<li>Divide the summations by each duration, and normalize them. As expressed in Table 2, for example, divide the summation for duration t = 0.0612 s by the duration 0.0612 s. The result is 3,351.5791. Then, divide the result by the total of the results in the fourth line in Table 2. The probability distribution is derived to be 0.64494.</li>
		<li>Plot the probability distributions for the bright events Pon(t) against their duration t in a logarithm-logarithm graph, and fit Log10Pon(t) by Log10(<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg" />) to deduce the power law exponent &#945;on for a specific blinking spot. If Pon(t) is fitted by <img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg" />, the fitted line deviates from the plots at small values of Pon(t), as shown by the dotted line in Figure 4A.</li>
		<li>Plot the probability distributions for dark events Poff(t) against their duration t in a logarithm-logarithm graph, and fit Log10Poff(t) by Log10(<img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg" /> to deduce the power law exponent &#945;off and the truncation time &#964;&#160;from the same blinking spot.&#160;If Poff(t) is fitted by <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg" />, the fitted curve deviates from the plots at small values of Poff(t).</li>
		<li>Repeat 3.2.1 to 3.2.8 for the other blinking spots in the video.</li>
	</ol>
	</li>
</ol>

<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><tbody><tr><td>Silver nitrate, 99.8%</td><td>Wako</td><td>194-00832</td><td></td></tr><tr><td>Trisodium citrate dihydrate, 99. %</td><td>Wako</td><td>191-01785</td><td></td></tr><tr><td>Poly-L-lysine aqueous solution, 0.1%</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>3,3&#039;-disulfopropylthiacyanine triethylamine</td><td>Hayashibara Biochemical Laboratories</td><td>NK-2703</td><td>a kind of thiacyanine dyes</td></tr><tr><td>3,3&#039;-diethyl-5,5&#039;-dichloro-9-methylthiacarbocyanine iodine salt</td><td>Hayashibara Biochemical Laboratories</td><td>SMP-9</td><td>a kind of thiacarobocyanine dyes</td></tr><tr><td>Sodium chloride, 99.5%</td><td>Wako</td><td>191-01665</td><td></td></tr><tr><td>Dimroth condenser</td><td>Iwaki</td><td>61-9722-22</td><td>perchased from AS ONE</td></tr><tr><td>Magnetic stirrer</td><td>Corning</td><td>DC-420D</td><td></td></tr><tr><td>Oil bath</td><td>Advantech</td><td>OS-220</td><td></td></tr><tr><td>Glass plate</td><td>Matsunami</td><td>S-1112</td><td>Microscope slide</td></tr><tr><td>Blower</td><td>Hozan</td><td>Z-288</td><td>Air duster</td></tr><tr><td>Liquid blocker pen</td><td>Daido Sangyo</td><td></td><td>LIQUID BLOCKER (Super Pap Pen). Ready-to-use hydrophobic barrier pen designed for immunohistochemistry applications</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-70</td><td></td></tr><tr><td>Objective lens</td><td>Olympus</td><td>LCPlanFl 60&amp;times;</td><td>NA 0.7</td></tr><tr><td>Dark field condenser</td><td>Olympus</td><td>U-DCD</td><td>NA 0.8&amp;ndash;0.92</td></tr><tr><td>Cooled digital CCD camera</td><td>Hamamatsu</td><td>ORCA-AG</td><td>controlled by software Aqua Cosmos</td></tr><tr><td>Software for the cooled digital CCD camera</td><td>Hamamatsu</td><td>AquaCosmos</td><td>used for also derivation of the time-profiles from the blinking spots in the video&amp;nbsp;</td></tr><tr><td>Color CCD camera</td><td>ELMO</td><td>TNC-C920</td><td>not used for analysis</td></tr><tr><td>DPSS laser</td><td>RGB laser system</td><td>NovaPro532-75</td><td>&amp;lambda; = 532 nm;&lt;br/&gt; 60 mW (corresponds to a power density of 600 W/cm2)</td></tr><tr><td>Interference filter</td><td>Semrock</td><td>LL01-532-12.5</td><td></td></tr><tr><td>Long pass filter</td><td>Semrock</td><td>BLP01-532R-25</td><td></td></tr><tr><td>Software for the distinguishment and counting of the bright/dark events</td><td>home-maid</td><td></td><td>programmed by C++</td></tr><tr><td>Software for the fitting by a power law</td><td>LightStone</td><td>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,...

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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 ...

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7.3K Views.  Kwansei Gakuin University. 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.

Video: Observation and Analysis of Blinking Surface-enhanced Raman Scattering

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 ...

Bioengineering

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Engineering

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, ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging technologies. Hyperspectral SRS and CARS imaging offer Raman spectral information at every pixel, which enables better separation of different chemical compositions. Although both techniques require two excitation lasers, their signal detection schemes and spectral properties are quite different. The goal of this protocol is to perform both hyperspectral SRS and CARS imaging on a single platform and compare the two microscopy techniques for imaging different biological samples. The spectral focusing method is employed to acquire spectral information using femtosecond lasers. By using standard chemical samples, the sensitivity, spatial resolution, and spectral resolution of SRS and CARS in the same excitation conditions (i.e., power at the sample, pixel dwell time, objective lens, pulse energy) are compared. The imaging contrasts of CARS and SRS for biological samples are juxtaposed and compared. The direct comparison of CARS and SRS performances would allow for optimal selection of the modality for chemical imaging.

This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity.

Stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) microscopy are the most widely used coherent Raman scattering imaging ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

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Rozdziały w tym wideo

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

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

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy