这项工作是部分由托斯卡纳大区和欧洲共同体的ERANET +项目LUS泡沫和BI-TRE的框架内支持。

注:金纳米棒的全部浓度是名义上金摩尔浓度表示。为与其它工作相比较，注意，1M的金大致相当于20μM的金纳米棒，在我们的情况。 1.阳离子金纳米棒的制备注意:该方法开始于西曲溴铵（CTAB）加帽的金纳米棒通过用抗坏血酸自催化还原氯金酸4的合成，根据根据Ratto 等人 26通过Nikoobakht 等 25介绍，并适于协议。然后这些金纳米棒是为了获得更多的生物相容性和亲和性血浆膜，由聚乙二醇的组合改性导线束10，11，27，28和季铵化合物22。 <ol><li>净化24毫升CTAB皑皑的金纳米棒在concentratio通过离心的两个周期（12,000 xg离心，30分钟）和倾析450微米的Au的n个。确保死体积至初始体积的比例大约是1/200或更低在这一协议的所有的离心步骤。使用500μM的水溶液CTAB作为洗涤溶液，并在含有500μM的CTAB和0.005％（体积/体积）聚山梨酯20的pH 5的颗粒最后转移到6毫升的100mM乙酸盐缓冲液。 </li><li>添加30微升10毫摩尔含水的α-甲氧基ω-巯基聚（乙二醇）（MW〜5000），并离开在37℃下30分钟以发生反应。 </li><li>添加30微升100mM（11-Mercaptoundecyl） - N，N，N-三甲基溴化物在二甲基亚砜和静止在37℃下放置24小时。 </li><li>接着，通过离心分离的四个周期（12,000 xg离心，30分钟）和倾析的水加18毫升0.005％（体积/体积）聚山梨醇酯20和纯化这些颗粒。在水使用0.005％（体积/体积）聚山梨醇酯20作为洗涤溶液并在pH颗粒最后转移到2.4毫升无菌PBS7.4。黄金最终标称浓度为4.5毫米。 </li></ol> 2.小​​鼠巨噬细胞的装载与金纳米棒<ol><li>使用单核细胞/巨噬细胞细胞系J774A.1和DMEM补充有10％胎牛血清，1 2mM谷氨酰胺，100单位/ ml青霉素和100μg/ ml链霉素的培养基。板5×10 5个细胞在60mm直径四个培养皿中，并允许他们生长24小时，以便在分离的时间被亚汇合（见步骤2.2）。 <ol><li>整个协议，保持标准培养条件下（37℃，5％CO 2，95％空气和100％相对湿度）下的细胞。使用层流柜和适当的个人防护装备操纵细胞。 </li></ol></li><li> 24小时后，装入阳离子金纳米棒的细胞，并准备他们被嵌入到壳聚糖膜: <ol><li>为了让由细胞吸收的颗粒中，添加4.5等分试样毫凹阳离子金纳米棒在PBS到每个培养皿，从而达到100μM的Au中的最终浓度。留在孵化培养皿24小时。 </li><li>接着，观察在光学显微镜下的细胞以确认其良好的条件和上载。细胞应表现出自己的正常形态和一些深色的细胞内囊泡。用刮刀分离细胞，合并那些来自两个培养皿中，以实现细胞的合适量为以下步骤（至少2×10 6个细胞），并离心它们（120 XG，6分钟），以消除任何多余的阳离子金纳米棒。该细胞团看起来应该几乎是黑色的。 </li><li>暂停于2ml PBS中沉淀并通过使用Bürker室的细胞计数。离心机含有2×10 6个细胞（120 XG，6分钟），并固定其沉淀在2ml 3.6％（重量/体积）在PBS中的甲醛在室温下十分钟悬浮液。最后，通过centrifugat洗此沉淀三次离子（120 XG，6分钟）以除去固定液。用PBS洗涤液。 </li></ol></li></ol> 3.埋入巨噬细胞的成壳聚糖膜注:壳聚糖26，27，28的特殊属性，29用来生产含阳离子金纳米棒染色的巨噬细胞仿生幻影。相对于其它的水凝胶诸如琼脂糖，壳聚糖使薄膜是强得多和更薄，这是用于PA显微镜6的关键。这些体模的制造是按照以前的协议29，30，31与作为规定在以下30一些修改进行。 <ol><li>制备酸化（pH 4.5，通过加入乙酸获得）和粘性3％（重量/体积）的低分子量脱乙酰壳多糖（平均分子量120 kDa）的溶液，充分混合，并让它在40℃均化24小时。 </li><li>接着，混合含阳离子金纳米棒（2×10 6个细胞）用500μl壳聚糖溶液的鼠巨噬细胞。 </li><li>为了获得〜50微米厚的幻影，倒入250毫克混合物的成1.91厘米2聚苯乙烯模具和离开他们在氮气流下24小时。此后，将这些样品与500微升的1M NaOH，以诱导交联，并用10ml超纯水的冲洗。 </li></ol> 4.光声转换的稳定性测试注:PA转化的稳定性由在参考文献6中描述的PA实验用自制的设置装置的影响。 <ol><li>通过使用塑料夹持器浸渍在培养皿的暂停含有在DI水中的巨噬细胞壳聚糖薄膜，例如，以保持从板的底面〜5mm的距离。将这个盘到微米级的XY工作台为了控制样本位置。 </li><li>聚焦用〜5纳秒脉冲持续时间的激光束在共振与金纳米棒（ 例如纵向电浆频带，从通过Q开关Nd的三次谐波泵浦光参量振荡器:YAG激光波长为400 - 2500 5纳秒的纳米和脉冲持续时间）垂直于与〜300微米的光斑直径在膜表面。 <ol><li>放置在激光出射前方的衰减器来调谐激光能量密度，并使用分束器来聚焦激光束的一部分，以能量计（ 例如 ，热电检测器）和监测注量的波动。保持对准期间低于〜光能量密度1兆焦耳/ 平方厘米每脉冲。每当激光是使用合适的激光护目镜。 </li></ol></li><li>浸超声换能器（1频率范围 - 20兆赫）到培养皿〜2毫米离膜表面的，并通过使用测微翻译调节其位置和旋转阶段最大限度地从电影发出的声信号。 </li><li>确定的探针通量楼罗不会损坏样品6: <ol><li>在约1毫焦/厘米2每脉冲，至少500次脉冲的注量照射样品的随机点。对于每一个脉冲，收购超声换能器和激光能量密度从示波器电能表相应的声信号。命名平均能量密度为F LO 的审判 。 </li><li>计算PA的信号作为其峰 - 峰振幅为脉冲数的函数的强度。为了配重的激光强度的波动，正常化每个声信号自身注量至F LO 试验的比率的幅度。分析的归一化的PA的强度作为脉冲数的函数的趋势并验证其随时间的稳定性。 </li><li>在不稳定的情况下，重复步骤4.4.1具有F LO的较低值至4.4.3 <s达&gt;审判。在稳定的情况下，重复使用F LO 试验的值由〜10％以上，直到稳定性的损失就出现了。设置探头通量楼罗为确保稳定性楼罗 审判的第二高值。 </li></ol></li><li>衡量一个重塑的阈值能量密度: <ol><li>选择样本的另一随机点以上以F LO 500的脉冲探测的平均PA强度（I LO 一个 ）。 </li><li>设置名义通量除了F LO更大，并提供50个脉冲。命名他们的平均能量密度为F EXC。 </li><li>再次探测，平均PA的强度（I LO 二 ）在以F LO 500脉冲。计算比R = I LO B / I LO 一个 。下面统一为R的值给出了样品的光学性质的不可逆的变化的证据。 </li><li>使用测微XY工作台移动薄膜，改变POIN随机样本吨。重复步骤4.5.1使用F EXC不同的值4.5.4，以便采取几个值的R的下方，左右及以上的统一。 15分是合理的。 </li><li>情节ř为F EXC的函数，并确定第作为该值的整形阈通量˚F当R开始从一个超越统计不确定性不同。6 </li></ol></li></ol>

作者宣称，他们没有竞争的经济利益。

靶向肿瘤相关巨噬细胞的概念正在成为一个强大的概念，以打击癌症34，35，36。在这里，而不是销毁，这些细胞被招募为移动车辆，使金纳米棒成肿瘤，其取向的剥削。这个角度看，需要颗粒的贴心设计，融入细胞及其鉴定。我们已发现，装载有阳离子金纳米棒的鼠巨噬细胞的耐光不会从颗粒的内吞囊泡内的限制，这意味着他们的电浆耦合和交叉过热不是...

在过去十年中，各种电浆颗粒如金纳米棒，纳米壳和纳米笼，在生物医学光学1，2，3，4已受到相当关注的应用。在与标准的金纳米球方差，这些较新的粒子共振在于提供用于通过主体最深光学穿透并用内源性组分1最高光学对比度的近红外（NIR）窗口。这个特性已引起对创新应用，例如光声（PA）的成像和癌症的光热消融的兴趣。然而，有几个问题限制这些颗粒的临床渗透。例如，它们的光学活性趋于诱导它们过热和修改他们的功能性形状朝向多个球形轮廓，其驱动一个光稳定性5，6，7，8 </SUP&gt; 9。占主导地位的科学辩论的另一个问题是他们的全身输送到肿瘤。尤其是金纳米棒相结合，是理想渗透了该显示增强通透性和保留和易用性恶性肿瘤标志物的特异性探针结合的肿瘤大小。因此，他们对直接注射到血流中制备被认为是一个可行的方案10，11，12，13。然而，这条路线仍然是个问题，其中大部分粒子变得由单核吞噬细胞系统10，11，12捕获。此外，另一个问题是，通过主体14循环后的粒子的光学和生化稳定性。当颗粒失去胶体稳定性和聚集，其等离子的特点和传热动态可能从电浆子耦合15受苦， </s达&gt; 16，17和交叉过热18。 最近，利用肿瘤相关巨噬细胞的趋性的概念已经成为一个聪明的替代19，20，21。这些细胞持有检测和高特异性的肿瘤普遍存在一种与生俱来的能力。因此，一个角度看可能是这些细胞从病人隔离， 与体外金纳米棒装载它们，然后注入回到患者，以意向使用它们作为负责递送的细胞的车辆。另一个优点是获得对颗粒的光学和生化稳定性更多的控制，因为它们的生物界面将在体外构建。尽管如此，这些细胞汽车作为光造影剂的性能需要一个批判性的分析。 在这项工作中，我们描述了准备和cellul的关键问题含有金纳米棒为癌症的PA的成像芳车辆。 PEG化的金纳米棒被修改与季铵化合物22，以实现该预期以促进与血浆膜23,24及其相互作用的阳离子分布。这些颗粒进行有效和非特异性摄取大部分细胞种类，希望不受外界干扰太多与他们的生物学功能。小鼠巨噬细胞装载有高达每细胞多达200，000阳离子金纳米棒，这成为紧内吞囊泡内密闭。这种配置应该出现的问题，因为这些囊泡内电浆耦合和交叉过热的威胁。因此，巨噬细胞被嵌入模仿生物组织生物聚合物水凝胶，以验证大多数颗粒PA转化的稳定性被保持在从生长培养基转移到内吞囊泡。有效实现了È测量标准，以便测量的PA转化为PA的成像直接兴趣的条件下的稳定性制定。一个重塑阈值设置在光不稳定性的非常发病50的激光脉冲10赫兹的典型重复率一个序列后。 我们相信，这些结果可能为新的发展战略，以实现等离子粒子肿瘤提供动力。

<table border="0" cellpadding="0" cellspacing="0" width="760">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hexadecyltrimethylammonium bromide</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">H6269</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gold(III) chloride trihydrate</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">520918</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silver nitrate</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">S6506</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-ascorbic acid</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5960</td>
			<td style="width:321px;">To synthesize gold nanorods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium borohydride</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:321px;">To synthesize gold nanoseeds</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MeO-PEG-SH</td>
			<td style="width:104px;">Iris Biotech</td>
			<td style="width:119px;">PEG1171</td>
			<td style="width:321px;">To PEGylate gold nanorods. Molecular weight about 5,000 Da</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acetic acid</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">320099</td>
			<td style="width:321px;">To PEGylate gold nanorods and solubilize chitosan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8750</td>
			<td style="width:321px;">To PEGylate gold nanorods</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">(11-Mercaptoundecyl)-N,N,N-trimethylammonium bromide</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">733305</td>
			<td style="width:321px;">To modify gold nanorods with quaternary ammonium compounds</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">276855</td>
			<td style="width:321px;">To solubilize (11-mercaptoundecyl)-N,N,N-trimethylammonium bromide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;&#160;Polysorbate 20</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2287</td>
			<td style="width:321px;">To centrifuge PEGylated gold nanorods</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">BE17-516F</td>
			<td style="width:321px;">To suspend gold nanorods before incubation with cells and to treat pellets of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">J774a.1</td>
			<td style="width:104px;">ATCC</td>
			<td style="width:119px;">TIB-67</td>
			<td style="width:321px;">Monocyte/macrophage murine cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">BE12-707F</td>
			<td style="width:321px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">DE14-801F</td>
			<td style="width:321px;">To be added to cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">BE17-605E</td>
			<td style="width:321px;">To be added to cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/streptomycin</td>
			<td style="width:104px;">Lonza</td>
			<td style="width:119px;">DE17-602E</td>
			<td style="width:321px;">To be added to cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:104px;">NEST</td>
			<td style="width:119px;">705001</td>
			<td style="width:321px;">Cell culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell scraper</td>
			<td style="width:104px;">EuroClone</td>
			<td style="width:119px;">ES7018</td>
			<td style="width:321px;">To detach cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formaldehyde</td>
			<td style="width:104px;">Fluka</td>
			<td style="width:119px;">47630</td>
			<td style="width:321px;">To fix cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chitosan, low molecular weight&#160;</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">448869</td>
			<td style="width:321px;">75-85% deacetylated. Molecular weight about 120,000 Da</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium hydroxyde</td>
			<td style="width:104px;">Sigma-Aldrich</td>
			<td style="width:119px;">306576</td>
			<td style="width:321px;">To insolubilize chitosan and generate the hydrogel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polystyrene cell culture plates</td>
			<td style="width:104px;">NEST</td>
			<td style="width:119px;">702011</td>
			<td style="width:321px;">Used as molds to fabricate chitosan hydrogels</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Optical parametric oscillator pumped by the third harmonic of a Q-switched Nd:YAG laser</td>
			<td style="width:104px;">Continuum, Santa Clara, USA</td>
			<td style="width:119px;">&#160;Surelite OPO plus</td>
			<td style="width:321px;">Source of optical excitation for photoacoustic tests</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pyroelectric detector&#160;</td>
			<td style="width:104px;">Gentec, Quebec, Canada</td>
			<td style="width:119px;">QE8SP</td>
			<td style="width:321px;">To monitor optical fluence for photoacoustic tests</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Pre amplified needle hydrophone</td>
			<td style="width:104px;">Precision Acoustic, Dorset, UK</td>
			<td style="width:119px;">Model with 1 mm sensor diameter and 1-20 MHz frequency range</td>
			<td style="width:321px;">To measure photoacoustic signals</td>
		</tr>
	</tbody>
</table>

preparation and photoacoustic analysis of cellular vehicles containing gold nanorods

金纳米棒是为一系列的生物医学应用，如光热消融和癌症的光声成像的吸引力，这要归功于在近红外窗口，低细胞毒性和潜在的对置成肿瘤其强烈的光学吸光度。然而，他们交付的肿瘤仍然是一个问题。一种创新的方法包括可装在体外金纳米棒肿瘤相关巨噬细胞的向性的开采。在这里，我们描述的制备方法和含有金纳米棒的细胞车辆的光声检查。 PEG化的金纳米棒被修改与季铵化合物，以实现一种阳离子轮廓。与普通培养皿小鼠巨噬细胞的接触，这些粒子被发现发生巨大摄取进入内吞囊泡。然后将这些细胞被包埋在生物聚合物水凝胶，这是用来验证光声转换的稳定性的颗粒被保持在其纳入蜂窝车辆。我们相信，这些结果可能为新型的发展战略提供等离子粒子肿瘤新的灵感。

这里，含有金纳米棒为癌症的PA的成像蜂窝车辆的可行性与协议的典型的结果一起示出。 图1中的TEM图像显示步骤1之后的颗粒和它们的细胞车辆步骤2.颗粒和细胞的TEM成像的制备别处17描述后的通常外观。阳离子金纳米棒进行巨噬细胞中一个巨大的积累，维持其正常形态。被发现的粒子被严密囊泡内吞局限?...

Watch this Scientific Journal Video about Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods at JoVE.com

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

preparation-photoacoustic-analysis-cellular-vehicles-containing-gold

Research

JoVE Journal

Bioengineering

6.8K 次观看.  Italian National Research Council. We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

视频: Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

微组织使用胶原蛋白的模块的制备凝胶含有细胞

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

使用光声血流循环黑色素瘤细胞的检测和隔离

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

集成光声眼底镜检查和谱域光学相干断层扫描

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

光声膀胱造影

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

含有宏观结构的光学组织幻影的制备与表征

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...