Yazar Prof. Y. Ozaki (Kwansei Gakuin Üniversitesi) ve Dr. T. Itoh (Ulusal Enstitüsü gelişmiş sanayi bilim ve teknoloji) Bu eser verimli onların tartışma için teşekkürler. Bu eser Milli Eğitim Bakanlığı, kültür, spor, bilim ve Teknoloji (No. 16 K 05671) KAKENHI (Grant-in-Aid için bilimsel araştırma C) tarafından desteklenmiştir.

1. numune hazırlama
<ol><li>
Gümüş kolloidal nano tanecikleri hazırlanması 20<ol>
<li>Gümüş kolloidal nano tanecikleri imal etmek 0,030 g gümüş nitrat ve TRISODYUM sitrat dihydrate 200 mL yuvarlak alt şişe su 150 ml 0,030 g geçiyoruz.</li>
		<li>Şişeye reflü (Dimroth) kondansatör ile birleştirin.</li>
		<li>Çözüm bir manyetik karıştırıcı ve heyecan çubuklu şişeye ilave edin. Sonra ısı bir yağ banyosu için 60 dk 150 ° C'de şişeye karıştırma çözümde. Not: Çözüm sarı, sonra sütlü gri dönecek.</li>
		<li>Oda sıcaklığında süspansiyon serin ve bir buzdolabı alüminyum folyo ile kaplı şişeye süspansiyon tutun. Not: Protokol bu noktada duraklatılmış. Kolloidal nano tanecikleri, bir buzdolabı, bir ay içinde sakladıktan sonra kullanın.</li>
	</ol>
</li>
	<li>
Çok renkli için örnek hazırlanması emisyon yanıp sönen 11<ol>
<li>Bir mikroskop slayt hazır için el ile bir cam levha sabun ile yıkayın ve su ile durulayın.</li>
		<li>Cam levha % 0,1 Poli-L-lizin sulu çözüm ekleyip çözüm bir üfleyici ile kaldırabilirsiniz.</li>
		<li>Cam levha gümüş kolloidal süspansiyon ekleyip süspansiyon körük ile kaldırabilirsiniz.</li>
		<li>Bırakma alanı cam plaka üzerinde bir sıvı engelleyici kalemle alın.</li>
		<li>Distile su cam tabakta bırakın ve bu mikroskop slayt oluşturmak ve su buharlaştırma gelen önlemek için başka bir cam levha ile kapsar.</li>
	</ol>
</li>
	<li>
Monoton renkli yanıp sönen SERS için örnek hazırlanması 7 , 8 , 9 , 10<ol>
<li>Bir mikroskop slayt hazır için el ile bir cam levha sabun ile yıkayın ve su ile durulayın.</li>
		<li>Gümüş kolloidal süspansiyon thiacyanine veya thiacarbocyanine boya ile karıştırın (25 veya 4 µM, sırasıyla) ve NaCl (10 mM) sulu çözüm adlı bir hacim oranı 2:1:1.</li>
		<li>Örnek süspansiyon cam levha üzerine bırakın ve süspansiyon bir üfleyici ile kaldırın.</li>
		<li>Bırakma alanı cam plaka üzerinde bir sıvı engelleyici kalemle alın.</li>
		<li>NaCl (1 M) sulu bir çözüm gümüş nano tanecikleri hareketsiz için cam tabakta bırakın ve bu bir mikroskop slayt tabak oluşturmak ve çözüm buharlaşan gelen önlemek için başka bir cam levha ile kapsar.</li>
	</ol>
</li>
</ol>2. gözlem gümüş nano tanecikleri yanıp sönen bir
<ol><li>
Örnek aydınlatma
	<ol>
<li>Protokolü 1.2 veya 1.3 ters bir mikroskop sahneye kullanılarak hazırlanan örnek cam levha yerleştirin.</li>
		<li>Beyaz ışık ile karanlık alan Kondenser ve odak bir objektif lens (60 X) kullanarak cam plaka üzerinde çeşitli renkli noktalar (mavi, yeşil, sarı ve kırmızı) kullanarak örnek cam levha aydınlatmak.</li>
		<li>Örnek yüzey göre 30 ° açılı Diod pompalı katı hal (DPSS) sürekli (cw) lazer bir girişim filtre aracılığıyla teslim zayıflatılmış bir ışını kullanarak örnek cam levha aydınlatmak.</li>
		<li>Aynı renkli çevreleyen içinde monoton renkli noktalar olarak gümüş nanoaggregates gözlemlemek için lazer aydınlatma kullanmak için lazer ışık alan görünüm merkezine taşımak ve z-yönde sahne ayarlayarak cam levha üzerindeki noktalar odaklanmak.</li>
	</ol>
</li>
	<li>
Yanıp sönen bir gözlem
	<ol>
<li>Uzun geçiren Filtre sonra objektif lens takın ve örnek yüzey göre 30 ° açılı bir müdahale süzgeci teslim DPSS cw-lazer ışını kullanarak örnek cam levha aydınlatmak.</li>
		<li>Belgili tanımlık kırpmak şekil 1 ' de gösterildiği gibi noktalar bulmak (ek malzeme Şekil S1 Ayrıca bakınız) tarafından sahne x - ve y-yönlere hareket ediyor.</li>
		<li>61-120 ms zaman çözünürlüğe sahip, soğutmalı dijital şarj kuplajlı cihaz (CCD) fotoğraf makinesi 20 dk için birleştiğinde ters mikroskop ile yanıp sönen noktalar video kaydetmek.</li>
	</ol>
</li>
</ol>3. SERS yanıp sönen analizi
<ol><li>
Zaman profil videodan türetme
	<ol>
<li>CCD kamera denetleyen yazılım, video dosyasını açın.</li>
		<li>Yanıp sönen noktalar ve karanlık alan seçmek için ayrı ayrı bölgeler ve video görüntüsü noktalar olmadan kapak alanları sürükleyin.</li>
		<li>Yanıp sönen noktalar ve karanlık yerlerde video sinyal yoğunluğu zaman profilleri türetmek için Zamansal analizi analizseçin ve Zamansal analizi penceresinde Calculate düğmesini tıklayın.</li>
		<li>Verileri bir metin dosyası olarak kaydedin.</li>
	</ol>
</li>
	<li>
Zaman profil analizi
	<ol>
<li>Temel zaman profilinin karanlık alanı ve/veya montaj rakamlar 2A ve 2B'yigösterildiği gibi bir polinom fonksiyon ile zaman profilden çıkararak düzleştirin.</li>
		<li>Yaklaşık 2000 puan, bentemelve bir standart sapma σ, temel yoğunluklarını, rakamlar 2C ve 2Dgösterildiği gibi oluşan bir ortalama temel yoğunluk değerlendirin.</li>
		<li>Parlak olayları karanlık olaylar, bentemel + 3σ bir eşik değerinden daha büyük şiddetlerde kullanma ayırmak ve her olay süresi kaydetmek. Şekil 3' te, örneğin, 0 olay 3.5476 için kayıt olarak karanlık olay lar ( t süresi olan 3.5476 = s) ve 3.5476 olay 4.0981 için kayıt parlak olay olarak s ( t süresi olan 0.5505 = s). Tablo 1' de gösterilen yordamı yineleyin.</li>
		<li>Aydınlık ve karanlık olaylar her süresince, Tablo 2birinci ve ikinci satırları ifade edildiği şekilde saymak.</li>
		<li>Etkinlik süresi tkısa hariç her süre için olayları sayısı toplamı. Tablo 2ikinci ve üçüncü hatları ifade edildiği şekilde, örneğin, her süresi için olayları sayısı toplamı ( t için olayları dışında 0.0612 = s) olarak 41 + 18 + 9 +...; sonuç toplamı t için eşittir 0.1223 = s, Yani 103.</li>
		<li>Simgenin her süresine göre bölmek ve onları normalleştirmek. Tablo 2' de ifade edildiği şekilde, örneğin, toplam süresi t için bölmek 0.0612 = süresi 0.0612 s s. 3,351.5791 sonucudur. Daha sonra Tablo 2' deki dördüncü satırı sonuçlarda Toplam Sonuç bölün. Olasılık dağılımı 0.64494 olmak elde edilir.</li>
		<li>Belgili tanımlık parlak olaylar Püzerinde(t) logaritma-logaritma grafikteki onların süresi t karşı için olasılık dağılımları arsa ve oturum10Püzerinde(t) günlük10 tarafından uygun (<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">) belgili tanımlık güç hukuk üs αüzerinde belirli bir nokta yanıp sönen için anlamak için. Eğer Püzerinde(t) tarafından monte <img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg"> </img>, monte satır sapma Püzerinde(t), küçük değerlerinin, araziler üzerinden şekil 4Anoktalı çizgi tarafından gösterildiği gibi.</li>
		<li>Olasılık dağılımları karanlık olaylar Pkapalı(t) logaritma-logaritma grafikteki onların süresi t karşı arsa ve10Pkapalı(t) uygun günlük10() tarafından <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> güç hukuk üs αkapalı ve aynı yanıp sönen üzerinden kesilme süresi τ spot anlamak için. Eğer Pkapalı(t) tarafından monte <img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg"> </img>, monte eğrisi sapma Pkapalı(t) küçük değerlerinde araziler üzerinden.</li>
		<li>3.2.1 3.2.8 video diğer yanıp sönen noktalar için yineleyin.</li>
	</ol>
</li>
</ol>

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Yazar ifşa etmek hiçbir şey vardır.

Gümüş nanoaggregate Kavşağı, SERS duyulur. Böylece, sitrat anyon ile kaplıdır kolloidal nano tanecikleri yerine nanoaggregates hazırlamak gerekir. Gümüş toplamları oluşan Poli-L--NH3+ ve SERS kökenlidir, lizin, ek tarafından oluşturulan etkisini Tuzlama üzerinden veya Na+ özellikler Şekil S2 takıma giren malzemenin içinde gösterildiği gibi NaCl, dan. Ayrıca, geniş alan birçok noktaları aydınlatmak için odaklanmamış lazer ışını örnek y...

Yüzey gelişmiş Raman (SERS) saçılma son derece hassas Raman spektroskopisi noble metal yüzey var. Raman spektrumu keskin tepe pozisyonları, fonksiyonel gruplar molekülleri halinde titreşim modları ile temel alan moleküler yapısı hakkında ayrıntılı bilgi sağlar beri tek bir molekül bilgi metal bir yüzeye araştırılması SERS1,2,3kullanarak. Bir adsorbate tek molekül düzeyinde ile gümüş nanoaggregate1,2,3,4,5,6, yanıp sönen sinyal görülmektedir 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16ve spektrum dalgalanmalar1,2,3,4,5,6,7,8, 9,10,11,12,13,14. Yanıp sönen rastgele bir nanometre boyutunda gümüş nanoaggregate kavşağında gelişmiş bir elektromanyetik (EM) alanı içinde ve dışında hareket eden tek bir molekül tarafından indüklenen. Bu nedenle, yanıp sönen bir Poisson dağılımı SERS yoğunluklarda ve BI-analit2,3,17bir tekniği ile karşılaştırıldığında tek molekül algılama için basit kanıt olarak kabul edilir. Ancak, güçlü Ag yüzeyinde moleküler davranışa bağlı, yanıp sönen ve dalgalanan spektrum detaylı mekanizmaları hala tartışmalı.
Önceki çalışmalarda SERS yanıp sönen Difüzyon katsayısı ve konsantrasyon bir gelişmiş EM alanı12,13içinde ve dışında,14 hareketli moleküllerin hesaplayabilir otokorelasyon işlevi kullanılarak analiz edilmiştir . Ayrıca, istikrarsızlık toplam yoğunluk temsil eder, bir normalleştirilmiş Standart sapma puanı sinyal15saat profilinden elde edilmiştir. Ancak, bu analitik yaklaşımlar birkaç moleküllerin davranış dayalı. Buna ek olarak, SERS yanıp sönen bir süper kararlılık düşsel içinde tanımlanan16tek molekül davranış gelişmiş EM alanı olabilir. Ancak, bu teknikler gelişmiş bir Elektromanyetik alan sadece böyle parametrelerinde elde edebilirsiniz. Geniş bir aralıktaki (örneğin, içinde SERS yanıp sönen) tek bir molekül rasgele davranışını bir ortalama4,5,6,7,8 yerine bir güç yasa olarak temsil edilebilir ,9,10,11, bir tek yarı iletken kuantum nokta (QD)18,19için yanıp sönen Floresans benzer. Bir güç hukuk analiz4,5,6,7,8,9,10,11, moleküler davranışı kullanarak parlak durumda (Gelişmiş EM alanı) ve karanlık durumu10tahmin edilebilir; diğer bir deyişle, tüm gümüş yüzey üzerinde molekül davranışını tahmin edilebilir.
Bu teknik, gümüş kolloidal nanoaggregates kullanılan4,5,olan6,7,8,9,10,11. Bu nanoaggregates belirli dalga boylarında heyecanlı oldukları zaman güçlü gelişmiş elektromanyetik alanlar etkileyen çeşitli yerelleştirilmiş yüzey plasmon rezonans (LSPR) grup göster. Böylece, nano tanecikleri kolloidal süspansiyon ve bazı veriler mevcut SERS-aktif gümüş hemen elde edilebilir. Belirli boyut, şekil ve düzenlemeler var, basit nanoyapıların söz konusu olduğunda yanıp sönen SERS LSPR bağımlılığı diğer bağımlılıklara7gizlemek; Yani, iyi ya da kötü nanostructure LSPR için kullanılırsa, parametreler sabit olacak ve diğer bağımlılıklara bu nedenle gizlenir. Güç hukuk analiz yanıp sönen SERS gümüş kolloidal nanoaggregates4,5,6,7,8, , çeşitli bağımlılıklara keşfetmek için kullanılan 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

Gümüş nanoaggregate merkezinde tek bir molekül yüzey gelişmiş Raman (SERS) saçılma yanıp sönen görülmektedir. Burada bir iletişim kuralı SERS-aktif hazırlamak konusunda anlatılan gümüş nanoaggregate, yanıp sönen belirli noktalar video mikroskobik görüntü kaydetmek ve yanıp sönen istatistikleri çözümlemek. Bu analizde, bir güç yasa onların süresi göreli olarak parlak olaylar için olasılık dağılımları üretir. Karanlık olaylar için olasılık dağılımları bir Üstel fonksiyon ile güç yasa ile donatılmıştır. Güç hukuk parametrelerinin moleküler davranış aydınlık ve karanlık Birleşik Devletleri temsil eder. Rastgele yürüyüş modeli ve molekül tüm gümüş yüzey üzerinde hızını tahmin edilebilir. Bu ortalamalar, otokorelasyon işlevleri ve süper kararlılık SERS düşsel kullanırken bile tahmin etmek zordur. Yanıp sönen kökenleri bu analiz yöntemi yalnız tarafından teyit edilemez çünkü gelecekte, güç hukuku analizleri spektral Imaging ile birleştirilmelidir.

Poli-L-lizin 1.2 iletişim kuralı tarafından hazırlanan ile gümüş nanoaggregates Resim 111' de gösterildiği gibi çeşitli renklerde yanıp sönen noktalardan SERS ve yüzey gelişmiş Floresans gözlenir. Buna ek olarak, SERS monoton renkli yanıp sönen noktalardan için gümüş nanoaggregates Protokolü 1.37,8,9,<sup cla...

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

Bu protokol yüzey gelişmiş Raman yanıp sönen analizi açıklar nedeniyle güç yasaları kullanarak bir gümüş yüzey üzerinde tek bir molekül random kaldırımı saçılma.

Gözlem ve yüzey gelişmiş Raman saçılması yanıp sönen analizi

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 Görüntüleme.  Kwansei Gakuin University. Bu protokol yüzey gelişmiş Raman yanıp sönen analizi açıklar nedeniyle güç yasaları kullanarak bir gümüş yüzey üzerinde tek bir molekül random kaldırımı saçılma.

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.

Rezonans ve spontan Raman Microspectroscopy Floresan Arkaplan reddi

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

Biyomühendislik

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.

Yüzey EBL Fabrikasyon Nanostructured Alt Tabakalar Kullanma biyomoleküllerin Raman Spektroskopisi Algılama Geliştirilmiş

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

Mühendislik

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.

Raman Spektroskopisi kullanarak Hücreler ve Ekstrasellüler Matriks Temassız, Etiket ücretsiz izleme

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.

Yüksek hızda sürekli uyarılan Brillouin saçılma Spektrometre için malzeme analizi

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).

Stimululated Raman saçılma dayalı doğrusal olmayan mikroskop uygulanması

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.

Geniş Bant Uyarılmış Raman Saçılma Mikroskobuna Dayalı Multipleks Kimyasal Görüntüleme

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.

Kimyasal Görüntüleme için Hiperspektral Uyarılmış Raman Saçılması ve Tutarlı Anti-Stokes Raman Saçılma Mikroskobunun Doğrudan Karşılaştırılması

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

Kimya

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1 CH stretch vibration in lipids and the 2120 cm-1 CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

Çifte rezonans Tutarlı Anti-Stokes Raman saçılımı (CARS) Biyolojik Yapıların Diferansiyel Görüntüleme

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Biyoloji

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.

İn vivo Kombine İki Foton Floresan ve Stimüle Raman Saçılma Mikroskobu ile Biyolojik Dokuların Görüntülenmesi

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.

Doku Teşhisi için Fare Beyninin Gerçek Zamanlı, İki Renkli Uyarılmış Raman Saçılma Görüntülemesi

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