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

著者は、この作品の実りある議論するため尾崎幸洋教授 (関西学院大学)、伊藤哲夫博士 (国立科学研究所の高度な産業と技術) をありがちましょう。この作業は、教育省、文化、スポーツ、科学および技術 (第 16 K 05671) から科研費 (費補助金科学研究 C) によって支えられました。

1. サンプル準備
<ol><li>
銀コロイドのナノ粒子の作製20<ol>
<li>銀コロイドのナノ粒子を作製するには、0.030 g 硝酸銀と 0.030 g 200 mL 丸底フラスコに水 150 mL にクエン酸ナトリウム二水和物を溶かします。</li>
		<li>(ジムロート) 還流凝縮器とフラスコを組み合わせます。</li>
		<li>磁気スターラー、攪拌棒でフラスコ内でソリューションをかき混ぜます。60 分 150 ° C で油浴中でフラスコの攪拌溶液を熱し。 注: ソリューションは、黄色、乳白色、グレーになります。</li>
		<li>室内の温度の懸濁液を冷却し、冷蔵庫にアルミ箔で覆われたフラスコの懸濁液を維持します。 注: プロトコルをこの時点で一時停止できます。コロイド状ナノ粒子を使用して、1 ヶ月以内、冷蔵庫で保存した後。</li>
	</ol>
</li>
	<li>
マルチカラーのサンプルの調製点滅発光11<ol>
<li>顕微鏡のスライドを準備ができて、石鹸でガラス板を手で洗って水ですすいでください。</li>
		<li>ガラス板に 0.1% ポリ L リジン水溶液を追加し、送風機とソリューションを削除します。</li>
		<li>ガラス板に銀コロイド懸濁液を追加および送風機を懸濁液を削除します。</li>
		<li>液体ブロッカー ペンでガラス板のドロップ領域を囲みます。</li>
		<li>ガラス板に蒸留水をドロップし、顕微鏡スライドを作成し、水が蒸発するを防ぐ別のガラス板でそれをカバーします。</li>
	</ol>
</li>
	<li>
単調な色点滅 SERS のサンプルの調製7,8,9,10<ol>
<li>顕微鏡のスライドを準備ができて、石鹸でガラス板を手で洗って水ですすいでください。</li>
		<li>銀コロイド懸濁液の thiacyanine または thiacarbocyanine の色素を混ぜて (25 または 4 μ M、それぞれ)、塩化ナトリウム (10 mM) の水溶液 2 の容積比で: 1:1。</li>
		<li>ガラス プレート上にサンプル サスペンションをドロップし、送風機とサスペンションを削除します。</li>
		<li>液体ブロッカー ペンでガラス板のドロップ領域を囲みます。</li>
		<li>銀ナノ粒子を固定化するガラス板に塩化ナトリウム (1 M) の水溶液をドロップし、顕微鏡スライド プレートを作成し、ソリューションを蒸発から防ぐために、別のガラス板でそれをカバーします。</li>
	</ol>
</li>
</ol>2. 点滅銀ナノ粒子の観察
<ol><li>
サンプルの照明
	<ol>
<li>倒立顕微鏡のステージ上の 1.2 または 1.3 プロトコルを使用したサンプルのガラス プレートを配置します。</li>
		<li>様々 な色の斑点 (青、緑、黄、および赤) 対物レンズ (60 X) を使用してガラス板上に暗視野コンデンサーとフォーカスを白色光を使用してサンプルのガラス プレートを照らします。</li>
		<li>ダイオード励起固体 (DPSS) 連続波 (cw) レーザー干渉フィルターを通してから試料表面に対して 30 ° の角度で配信、弱毒ビームを用いたサンプル ガラス板を照らします。</li>
		<li>周辺の同じ色に単調な色の斑点として銀ハイパーラマンを観察するレーザ光を照射して、レーザー照射面積をビューの中央に移動、z 方向の段階を調整することによってガラス プレート上のスポットに焦点を当てます。</li>
	</ol>
</li>
	<li>
点滅の観察
	<ol>
<li>ロングパス ・ フィルターを対物レンズの後に挿入し、試料表面に対して 30 ° の角度で干渉フィルターを通して配信 DPSS cw レーザー光を用いたサンプル ガラス板を照らします。</li>
		<li>図 1に示すように、点滅してスポットを見つける (また見なさい図 S1補足資料で) x 方向と y 方向でステージを移動することによって。</li>
		<li>20 分間の 61-120 ms の時間分解能を持っている冷却デジタル電荷結合素子 (CCD) カメラに結合、倒立顕微鏡で点滅している地点のビデオを記録します。</li>
	</ol>
</li>
</ol>3 点滅表面増強ラマン散乱の解析
<ol><li>
時間プロフィール ビデオからの導出
	<ol>
<li>CCD カメラを制御するソフトウェア、ビデオのファイルを開きます。</li>
		<li>点滅スポット、暗い領域を選択すると、別にビデオ イメージのスポットとの領域をカバーする領域をドラッグします。</li>
		<li>点滅スポットやビデオの暗い部分から信号強度の時間分布を導出するには、分析、時系列解析を選択し、時系列解析] ウィンドウで[計算] をクリックします。</li>
		<li>テキスト ファイルとしてデータを保存します。</li>
	</ol>
</li>
	<li>
時間プロファイルの解析
	<ol>
<li>時間プロファイルのベースラインは、暗い領域および/または図 2 aと2 bに示すように、多項式関数でフィッティングから時間プロファイルを引いて平らにします。</li>
		<li>図 2と2 Dに示すように約 2000 点、私基本基準強度の標準偏差は、σ, から成る平均基準強度を評価します。</li>
		<li>私基本+ 3 σ、しきい値よりも大きい強度を使用してイベントを暗いから明るいイベントを区別し、各イベントの存続期間を記録します。図 3、たとえば、3.5476 を 0 からイベント記録暗いイベントとして s (持続時間t = 3.5476 s)、4.0981 に 3.5476 からイベントを記録および明るいイベントとして s (持続時間t = 0.5505 s)。表 1に示すように、手順を繰り返します。</li>
		<li>表 2の最初と 2 番目の行で表さ各期間の明るい部分と暗いイベントの数をカウントします。</li>
		<li>期間tよりも短い間隔でイベントを除いて、各期間のイベントの数を合計します。各期間のイベントの数をたとえば、合計表 2の 2 番目と 3 番目の行に示された、( tのイベントを除いて 0.0612 = s) 41 + 18 + 9 +...;その結果tの総和に等しい = 0.1223 s、すなわち103。</li>
		<li>各期間合計で割ります、正規化します。表 2に示された、たとえば、分割期間tの合計 = 0.0612 期間 0.0612 s で s。結果は 3,351.5791 です。次に、表 2の 4 行目の結果の合計で結果を分割します。確率分布は、0.64494 に派生します。</li>
		<li>明るいイベントP、上(t) 対数-対数グラフで自分の時間tに対して確率分布をプロットし、ログ10Pの(t)10 のログ(<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">)、電源法指数 αに特定のスポットで点滅を推測します。Pの(t) が取り付けられている場合<img alt="Equation 1" src="/files/ftp_upload/56729/56729eq1.jpg">、近似直線は、図 4Aの点線で示すように、 Pの(t) の小さな値でプロットから逸脱。</li>
		<li>暗いイベントPオフ(t) 対数-対数グラフではtの期間に対する確率分布をプロットし、ログ10(ログ10Pから(t) に合わせてください<img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg">電源法指数 α のオフと、切り捨て時間τ同じ点滅からスポットを推測します。Pオフ(t) が取り付けられている場合<img alt="Equation 3" src="/files/ftp_upload/56729/56729eq3.jpg">、 Pオフ(t) の小さな値でプロットから逸脱する近似曲線。</li>
		<li>ビデオで他の点滅しているスポットを 3.2.1 に 3.2.8 を繰り返します。</li>
	</ol>
</li>
</ol>

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著者は、何を開示します。

銀 nanoaggregate ジャンクションからセールが出力されます。したがって、我々 はコロイド状ナノ粒子、クエン酸アニオンで覆われているよりもむしろハイパーラマンを準備する必要。銀の集計は、ポリ-L-リジン、NH3+し、サールの起源の付加によって作成されるエフェクトを塩漬けから形成されているまたは塩化ナトリウム、補足資料の図 S2のようにから Na+</sup...

表面増強ラマン散乱 (SERS) は、機密性の高い貴金属表面ラマン分光です。金属表面上の単一分子の情報を調べることができますラマン スペクトル、分子内の機能グループの振動モードを鋭いピークの位置に基づく分子の構造に関する詳細情報を提供しますのでSERS1,2,3を使用しています。分子レベルで吸着と銀 nanoaggregate から点滅信号が観察される1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16、およびスペクトル変動1,2,3,4,5,6,7,8、 9,10、11,12,13,14。点滅は、ナノサイズの銀 nanoaggregate ジャンクションで強化された電磁 (EM) フィールドがランダムに出入りする単一分子誘起することができます。したがって、点滅と、表面増強ラマン散乱強度および bi 検体2,3,17のポアソン分布を用いた手法と比較して、単一分子検出のための簡単な証拠と見なされます。ただし、Ag 表面上の分子の挙動に依存が強く、点滅と変動のスペクトルの詳細なメカニズムはまだ論争を呼びます。
従来、SERS の点滅が分析されて拡散係数と、強化された EM フィールド12,13,14 出入りする分子の濃度を計算することができます自己相関関数を使用して.さらに、全磁力の不安定性を表す、正規化された標準偏差のスコアは、信号15時間プロファイルから派生しています。ただし、これらの分析方法は、いくつかの分子の挙動に基づく可能性があります。対照的に、SERS の点滅の超解像イメージング、強化された電磁界での単一分子の挙動は、識別された16をすることができます。ただし、これらのテクニックは、強化された EM 分野のみでこのようなパラメーターを取得できます。平均4,5,6,7,8 よりもむしろ力法律として (例えば、SERS の点滅) の広い範囲での単一分子のランダムな動作を表現できます。 ,9,10,11, 半導体単一量子ドット (QD)18,19から点滅蛍光に似ています。電力法分析4,5,6,7,8,9,10,11, 分子の挙動を使用して(強化された電磁界) で明るい状態と暗い状態10; の両方に推定することができます。つまり、全体の銀表面上の分子の挙動を推定できます。
この技は、銀コロイド ハイパーラマンが使用される4,5,6,7,8,9,10,11。これらハイパーラマンは、ある特定の波長で興奮しているとき強く強化された電磁界に影響を与える各種の局在表面プラズモン共鳴 (LSPR) バンドを示します。したがって、SERS 活性銀ナノ粒子コロイド懸濁液といくつかのデータに存在するがすぐに得られます。単純なナノ構造、ある特定のサイズ、形状、および手配の場合点滅 SERS の LSPR の依存性は、他の依存の7; を隠すことができます。すなわち、LSPR に良し悪しのナノ構造を使用する場合、パラメーターは定数になります、他の依存したがって隠されています。銀コロイド ハイパーラマン4,5,6,7,8,から点滅 SERS の様々 な依存症を発見するパワー法解析が使用されています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

銀 nanoaggregate 接合部での単一分子から点滅表面増強ラマン散乱 (SERS) が観察されます。SERS 活性を準備する方法のプロトコルを表示するここでは、銀の nanoaggregate、顕微鏡画像の特定の点滅しているスポットのビデオを記録し、点滅の統計情報を分析します。この分析では、べき乗則はその期間を基準にして明るいイベントの確率分布を再現します。暗いイベントの確率分布は、指数関数との力法律によって取り付けられています。べき乗則のパラメーターは、明るいと暗い状態での分子の挙動を表します。ランダムウォーク モデルおよび銀表面全体に分子の速度を見積もることができます。それは平均、自己相関関数、および超解像 SERS のイメージングを使用する場合でもを見積もるは困難です。将来は、点滅の起源は、単独でこの分析法による確認ができないため電力法解析スペクトル イメージングを組み合わせる必要があります。

プロトコル 1.2 により作製したポリ L リジンと銀のハイパーラマンからは、 1 図11に示すように SERS と表面増強蛍光から色とりどりの点滅しているスポットを観察します。対照的に、表面増強ラマン散乱から単調な色の点滅の斑点は、プロトコル 1.37,8,9,</...

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

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

このプロトコルを記述する点滅表面増強ラマン分析発電法を用いた銀表面の単一分子のランダム ・ ウォークによる散乱します。

点滅表面増強ラマン散乱の観測と解析

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

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7.3K ビュー.  Kwansei Gakuin University. このプロトコルを記述する点滅表面増強ラマン分析発電法を用いた銀表面の単一分子のランダム ・ ウォークによる散乱します。

ビデオ: 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.

共鳴と自発ラマン顕微の蛍光バックグラウンドの除去

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

生物工学

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.

表面EBL作製したナノ構造基板を用いた生体分子のラマン分光検出の強化

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

工学

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.

ラマン分光法を用いた細胞と細胞外マトリックスの非接触、ラベルフリーの監視

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.

材料の分析のための高速連続誘導ブリルアン散乱分光器

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).

刺激されたラマン散乱に基づく非線形顕微鏡の実施

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.

広帯域刺激ラマン散乱顕微鏡に基づく多重化学イメージング

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.

化学イメージングのためのハイパースペクトル刺激ラマン散乱とコヒーレントアンチストークスラマン散乱顕微鏡の直接比較

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

化学

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.

インビボ で2光子蛍光と誘導ラマン散乱顕微鏡を組み合わせた生体組織のイメージング

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.

組織診断のためのマウス脳のリアルタイム、2色刺激ラマン散乱イメージング

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.

刺激されたラマン散乱顕微鏡を用いた カエノラム型エレガンス における脂質蓄積ダイナミクスの無標識イメージング

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

点滅表面増強ラマン散乱の観測と解析

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共鳴と自発ラマン顕微の蛍光バックグラウンドの除去

表面EBL作製したナノ構造基板を用いた生体分子のラマン分光検出の強化

ラマン分光法を用いた細胞と細胞外マトリックスの非接触、ラベルフリーの監視

材料の分析のための高速連続誘導ブリルアン散乱分光器

刺激されたラマン散乱に基づく非線形顕微鏡の実施

広帯域刺激ラマン散乱顕微鏡に基づく多重化学イメージング

化学イメージングのためのハイパースペクトル刺激ラマン散乱とコヒーレントアンチストークスラマン散乱顕微鏡の直接比較

インビボで2光子蛍光と誘導ラマン散乱顕微鏡を組み合わせた生体組織のイメージング

組織診断のためのマウス脳のリアルタイム、2色刺激ラマン散乱イメージング

刺激されたラマン散乱顕微鏡を用いたカエノラム型エレガンスにおける脂質蓄積ダイナミクスの無標識イメージング

点滅表面増強ラマン散乱の観測と解析

要約

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共鳴と自発ラマン顕微の蛍光バックグラウンドの除去

表面EBL作製したナノ構造基板を用いた生体分子のラマン分光検出の強化

ラマン分光法を用いた細胞と細胞外マトリックスの非接触、ラベルフリーの監視

材料の分析のための高速連続誘導ブリルアン散乱分光器

刺激されたラマン散乱に基づく非線形顕微鏡の実施

広帯域刺激ラマン散乱顕微鏡に基づく多重化学イメージング

化学イメージングのためのハイパースペクトル刺激ラマン散乱とコヒーレントアンチストークスラマン散乱顕微鏡の直接比較

インビボ で2光子蛍光と誘導ラマン散乱顕微鏡を組み合わせた生体組織のイメージング

組織診断のためのマウス脳のリアルタイム、2色刺激ラマン散乱イメージング

刺激されたラマン散乱顕微鏡を用いた カエノラム型エレガンス における脂質蓄積ダイナミクスの無標識イメージング

インビボで2光子蛍光と誘導ラマン散乱顕微鏡を組み合わせた生体組織のイメージング

刺激されたラマン散乱顕微鏡を用いたカエノラム型エレガンスにおける脂質蓄積ダイナミクスの無標識イメージング