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

L'autore ringrazia il Prof Y. Ozaki (Kwansei Gakuin University) e Dr. T. Itoh (National Institute of Advanced Industrial Science and Technology) per la loro proficua discussione su quest'opera. Questo lavoro è stato supportato da KAKENHI (sovvenzione per Scientific Research C) dal Ministero della pubblica istruzione, cultura, sport, scienza e tecnologia (n. 16K 05671).

1. preparazione del campione
<ol><li>
Preparazione di nanoparticelle di argento colloidale 20<ol>
<li>Per fabbricare le nanoparticelle d'argento colloidale, sciogliere 0,030 g di nitrato d'argento e 0,030 g di citrato trisodico biidrato in 150 mL di acqua in un pallone a fondo tondo 200 mL.</li>
		<li>Combinare la beuta con un refrigerante a ricadere (Dimroth).</li>
		<li>Agitare la soluzione in un matraccio con una barra magnetica di agitatore e mescolare. Quindi, riscaldare la soluzione mescolando nel matraccio in bagno d'olio a 150 ° C per 60 min. Nota: La soluzione diventerà grigio giallo, poi latteo.</li>
		<li>Raffreddare la sospensione a temperatura ambiente e a mantenere la sospensione nel pallone coperto con foglio di alluminio in frigorifero. Nota: Il protocollo può essere sospesa a questo punto. Utilizzare le nanoparticelle colloidali, dopo la conservazione in frigorifero, entro un mese.</li>
	</ol>
</li>
	<li>
Preparazione del campione per multi-colorato lampeggiante emissione 11<ol>
<li>Per preparare un vetrino da microscopio, lavare a mano, una lastra di vetro con sapone e risciacquare con acqua.</li>
		<li>Aggiungere 0,1% soluzione acquosa di poli-L-lisina per la lastra di vetro e rimuovere la soluzione con un ventilatore.</li>
		<li>Aggiungere la sospensione colloidale d'argento per la lastra di vetro e rimuovere la sospensione con un ventilatore.</li>
		<li>Racchiudere un'area di rilascio sulla lastra di vetro con una penna di liquido bloccante.</li>
		<li>Acqua distillata goccia sulla lastra di vetro e coprire con un'altra lastra di vetro per creare un vetrino da microscopio e impedire l'evaporazione di acqua.</li>
	</ol>
</li>
	<li>
Preparazione del campione per monotono SERS lampeggiante colorato 7 , 8 , 9 , 10<ol>
<li>Per preparare un vetrino da microscopio, lavare a mano, una lastra di vetro con sapone e risciacquare con acqua.</li>
		<li>Mescolare la sospensione colloidale d'argento con la tintura di thiacyanine o thiacarbocyanine (25 o 4 µM, rispettivamente) e una soluzione acquosa di NaCl (10 mM) a un rapporto volumetrico di 2:1:1.</li>
		<li>Eliminare la sospensione del campione sulla piastra di vetro e rimuovere la sospensione con un ventilatore.</li>
		<li>Racchiudere un'area di rilascio sulla lastra di vetro con una penna di liquido bloccante.</li>
		<li>Goccia di una soluzione acquosa di NaCl (1 M) sul piatto di vetro per immobilizzare le nanoparticelle d'argento e coprire con un'altra lastra di vetro per creare una piastra di scorrimento microscopio e impedire che la soluzione di evaporazione.</li>
	</ol>
</li>
</ol>2. osservazione del lampeggiante nanoparticelle d'argento
<ol><li>
Illuminazione del campione
	<ol>
<li>Posizionare la piastra di vetro di campione preparata con protocollo 1.2 o 1.3 sulla scena di un microscopio invertito.</li>
		<li>Illuminare la lastra di vetro del campione usando la luce bianca attraversa un condensatore per campo oscuro e la messa a fuoco su vari punti colorati (blu, verde, gialli e rossi) sulla lastra di vetro utilizzando una lente obiettiva (60 X).</li>
		<li>Illuminare la lastra di vetro di campione utilizzando un fascio di luce attenuata, consegnato ad un angolo di 30° rispetto alla superficie del campione, da un laser a diodi a stato solido pompato (DPSS) continuo-fluttui (cw) attraverso un filtro antidisturbo.</li>
		<li>Per utilizzare laser illuminazione per osservare l'argento nanoaggregati come macchie di colore monotoni in zona dello stesso colore, spostare l'area di illuminazione laser al centro della vista e concentrarsi sui punti sulla lastra di vetro regolando la fase nella direzione z.</li>
	</ol>
</li>
	<li>
Osservazione del lampeggiante
	<ol>
<li>Inserire un filtro passa-lungo dopo la lente dell'obiettivo e illuminare la lastra di vetro di campione utilizzando un fascio di cw-laser DPSS consegnato ad un angolo di 30° rispetto la superficie del campione attraverso un filtro antidisturbo.</li>
		<li>Trovare il lampeggio macchie come mostrato in Figura 1 (Vedi anche Figura S1 nel materiale supplementare) spostando il palco nelle direzioni x e y.</li>
		<li>Registrare video dei luoghi lampeggianti con il microscopio invertito, accoppiato ad una fotocamera raffreddato digitale charge coupled device (CCD), che ha una risoluzione temporale di 61-120 ms, per 20 min.</li>
	</ol>
</li>
</ol>3. analisi del lampeggiante SERS
<ol><li>
Derivazione del profilo di tempo dal video
	<ol>
<li>Nel software che controlla la telecamera CCD, aprire il file video.</li>
		<li>Per selezionare i punti lampeggianti e zona scura, trascinare aree che coprono separatamente aree con e senza macchie nell'immagine video.</li>
		<li>Per derivare i profili orario di intensità di segnale dal lampeggiante macchie e aree scure nel video, selezionare Analisi temporale nell'analisie fare clic su Calcola nella finestra Analisi temporale .</li>
		<li>Salvare i dati come file di testo.</li>
	</ol>
</li>
	<li>
Analisi del profilo tempo
	<ol>
<li>Appiattire una previsione del profilo orario sottraendo il profilo orario dall'area scura e/o raccordo con una funzione polinomiale, come mostrato nelle figure 2A e 2B.</li>
		<li>Valutare un'intensità media della linea di base che consiste di circa 2000 punti, iobasee una deviazione standard di intensità della linea di base, σ, come mostrato nelle figure 2 e 2D.</li>
		<li>Distinguere eventi luminosi da eventi oscuri con maggiore intensità di una soglia di hobase + 3 σ e registrare la durata di ogni evento. Nella Figura 3, ad esempio, registrare l'evento da 0 a 3.5476 s come l'evento scuro (con una durata di t = 3.5476 s) e registrare l'evento da 3.5476 a 4.0981 s come l'evento luminoso (con una durata di t = 0.5505 s). Ripetere la procedura come indicato nella tabella 1.</li>
		<li>Contare il numero di eventi di chiari e scuri per ogni durata, come espresso nelle linee prime e seconda della tabella 2.</li>
		<li>Sommare il numero di eventi per ogni durata, fatta eccezione per eventi più brevi di durata t. Come espresso nella seconda e terza riga della tabella 2, ad esempio, sommare il numero di eventi per ogni durata (fatta eccezione per gli eventi per t = 0.0612 s) come 41 + 18 + 9 +...; il risultato è uguale la somma per t = 0.1223 s, cioè 103.</li>
		<li>Dividere le sommatorie per ogni durata e normalizzarli. Come espresso nella tabella 2, per esempio, dividere la somma per durata t = 0.0612 s dalla durata 0.0612 s. Il risultato è 3,351.5791. Poi, dividere il risultato per il totale dei risultati nella quarta riga nella tabella 2. La distribuzione di probabilità è derivata per essere 0.64494.</li>
		<li>Tracciare le distribuzioni di probabilità per il brillante eventi Psu(t) contro la loro durata t in un grafico del logaritmo logaritmo e montare Log10Psu(t) di Log10 (<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">) per dedurre il power legge esponente αsu per uno specifico lampeggio spot. Se Psu(t) viene montata dal <img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg"> </img>, la linea componibile devia dai terreni a piccoli valori di Psu(t), come indicato dalla linea tratteggiata in Figura 4A.</li>
		<li>Tracciare le distribuzioni di probabilità per eventi oscuri Poff(t) contro la loro durata t in un grafico del logaritmo logaritmo e Log10Poff(t) in forma di Log10( <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> dedurre power legge esponente αfuori e il troncamento tempo τ dal lampeggio stesso spot. Se Poff(t) viene montata dal <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> </img>, la curva si discosta dai terreni a piccoli valori di Poff(t).</li>
		<li>Ripetere 3.2.1 a 3.2.8 per gli altri punti lampeggianti nel 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>

L'autore non ha nulla di divulgare.

Dallo svincolo di nanoaggregati argento, SERS è emessa. Quindi, abbiamo bisogno di preparare nanoaggregati piuttosto che nanoparticelle colloidali, che sono coperti con gli anioni citrato. Aggregati d'argento sono formati dall'effetto creato tramite l'aggiunta di poli-L-lisina, che ha -NH3+ ed è l'origine della SERS, la salatura o cationi Na+ da NaCl, come mostrato in Figura S2 del materiale supplementare. Inoltre, per illuminare i molti punti nella vasta area, il raggi...

Superficie-enhanced Raman scattering (SERS) è altamente sensibile spettroscopia Raman da una superficie di metallo nobile. Poiché lo spettro Raman fornisce informazioni dettagliate sulla struttura molecolare sulla base delle posizioni di picco acuto, attraverso i modi vibrazionali di gruppi funzionali nelle molecole, è possibile analizzare le informazioni di una singola molecola su una superficie metallica usando SERS1,2,3. Da un argento nanoaggregati con un adsorbato a livello di singola molecola, un segnale lampeggio è osservato1,2,3,4,5,6, 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16e lo spettro fluttua1,2,3,4,5,6,7,8, 9,10,11,12,13,14. Lampeggiante può essere indotta da una singola molecola che casualmente si muove dentro e fuori un maggiore campo elettromagnetico (EM) in un incrocio di nanoaggregati d'argento di dimensioni nanometriche. Di conseguenza, lampeggiante è considerato prova semplice per il rilevamento di singola molecola, confrontato con una tecnica che utilizza una distribuzione di Poisson di intensità di SERS e un bi-analita2,3,17. Tuttavia, i meccanismi dettagliati dello spettro lampeggio e fluttuante, che può dipendere fortemente il comportamento molecolare sulla superficie Ag, sono ancora controversi.
Negli studi precedenti, lampeggiante SERS è stato analizzato utilizzando la funzione di autocorrelazione, che può calcolare il coefficiente di diffusione e la concentrazione delle molecole in movimento dentro e fuori un'avanzata EM campo12,13,14 . Inoltre, un punteggio di deviazione standard normalizzata, che rappresenta instabilità dell'intensità totale, è stato derivato dal profilo tempo del segnale15. Tuttavia, questi approcci analitici possono basarsi sul comportamento di alcune molecole. Al contrario, in un imaging di Super-risoluzione del lampeggiante SERS, comportamento di singola molecola in un maggiore campo di EM può essere identificato16. Tuttavia, queste tecniche possono ottenere tali parametri solo in un maggiore campo di EM. Il comportamento casuale di una singola molecola all'interno di una vasta gamma (per esempio, nel lampeggiante SERS) può essere rappresentato come una legge di potenza piuttosto che una media4,5,6,7,8 ,9,10,11, simili a fluorescenza lampeggia da un singolo semiconductor quantum dot (QD)18,19. Utilizzando un potere legge analisi4,5,6,7,8,9,10,11, comportamento molecolare può essere stimato in stato brillante (nel campo EM avanzato) sia stato scuro10; ovvero, il comportamento della molecola su tutta la superficie d'argento può essere stimato.
Per questa tecnica, argento colloidale nanoaggregati sono usato4,5,6,7,8,9,10,11. Questi nanoaggregati mostrano varie bande di risonanza (LSPR) plasmonica di superficie localizzata che influiscono fortemente rafforzate campi elettromagnetici quando sono eccitati a determinate lunghezze d'onda. Così, argento SERS-attivo nanoparticelle presenti in sospensione colloidale e alcuni dati immediatamente possa essere ottenuto. Nel caso di semplice nanostrutture, che hanno accordi, forme e dimensioni specifiche, la dipendenza di LSPR della SERS lampeggiante può celare altre dependences7; vale a dire, se la nanostruttura buona o cattiva a LSPR viene utilizzata, i parametri sarà costanti, e le altre dipendenze pertanto saranno nascosto. Analisi della legge di potenza sono stato utilizzato per scoprire varie dipendenze della SERS lampeggiante da argento colloidale nanoaggregati4,5,6,7,8, 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

Da una singola molecola in un incrocio di nanoaggregati argento, lampeggiante superficie-enhanced Raman scattering (SERS) è osservato. Qui, un protocollo è presentato su come preparare il SERS-attivo argento nanoaggregati, registrare un video di alcuni punti lampeggianti dell'immagine microscopica e analizzare le statistiche di lampeggiante. In questa analisi, una legge di potenza riproduce le distribuzioni di probabilità per eventi luminosi rispetto alla loro durata. Le distribuzioni di probabilità per eventi oscuri sono dotate di una legge di potenza con una funzione esponenziale. I parametri della legge di potere rappresentano il comportamento molecolare negli Stati sia chiaro e scuri. Il modello di random walk e la velocità della molecola su tutta la superficie d'argento può essere stimati. È difficile stimare anche quando si utilizzano medie, funzioni di autocorrelazione e super-risoluzione SERS imaging. In futuro, analisi di legge di potenza dovrebbero combinarsi con imaging spettrale, perché le origini del lampeggiante non possono essere confermate da questo metodo di analisi da solo.

Dall'argento nanoaggregati con poli-L-lisina preparato dal protocollo 1.2, si osservano macchie multicolore lampeggiante da SERS e superficie-enhanced fluorescenza, come illustrato nella Figura 111. Al contrario, monotoni punti colorati lampeggiante da SERS sono stati osservati per i nanoaggregati d'argento con le molecole di colorante preparate dal protocollo 1.37,8,</sup...

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

Questo protocollo descrive l'analisi del lampeggiante superficie-enhanced Raman scattering dovuto il cammino casuale di una singola molecola su una superficie d'argento utilizzando leggi di potenza.

Osservazione e analisi del lampeggiante superficie-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 ...

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

7.4K Views.  Kwansei Gakuin University. Questo protocollo descrive l'analisi del lampeggiante superficie-enhanced Raman scattering dovuto il cammino casuale di una singola molecola su una superficie d'argento utilizzando leggi di potenza.

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.

Rifiuto di fluorescenza di fondo in risonanza e spontanea microspettroscopia Raman

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

Ricerca

Bioingegneria

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.

Surface Enhanced Raman Spectroscopy Rilevamento di Biomolecole Utilizzando EBL manufatti nanostrutturati Substrati

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

Ingegneria

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.

Senza contatto, Label-free monitoraggio delle cellule e matrice extracellulare mediante Spettroscopia Raman

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.

Ad alta velocità continuo-fluttui stimolato Brillouin Scattering spettrometro per analisi materiali

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).

Implementazione di un microscopio non lineare basato sullo scattering Raman stimolato

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.

Imaging chimico multiplex basato sulla microscopia a dispersione Raman stimolata a banda larga

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.

Confronto diretto tra lo scattering Raman stimolato iperspettrale e la microscopia a dispersione raman coerente anti-Stokes per l'imaging chimico

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

Chimica

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.

In vivo Imaging di tessuti biologici con fluorescenza combinata a due fotoni e microscopia a dispersione Raman stimolata

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.

In tempo reale, a due colori stimolato Raman Scattering Imaging del cervello di topo per la diagnosi dei tessuti

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.

Imaging privo di etichette delle dinamiche di conservazione lipidica negli elegani di caenorhabditis utilizzando la microscopia a dispersione Raman stimolata

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