作者要感谢新加坡教育部（ARC2 / 15:M4020238）资助的二级拨款的财政支持。作者还要感谢周伟荣博比机车店的帮助。

所有动物实验均按照新加坡南洋理工大学机构动物保护和使用委员会（动物标本编号ARF-SBS / NIE-A0263）的批准规定和指导方针进行。 AR-OR-PAM系统（ 图1 ） <ol><li> 系统配置:AR-PAM <ol><li>使用由二极管泵浦的固态Nd-YAG激光器（532nm）和可调谐范围为559-576nm的染料激光器组成的纳秒可调谐激光器系统作为光学照射源。使用外部控制器将激光波长设置为570 nm，使用激光软件将激光重复率设置为1 kHz。 </li><li>在激光前面放置45°角的光束采样器，通过可变中性密度滤光片（NDF1; OD = 0-4.0）将5％的激光功率转移到光电二极管。 </li><li>使用光束采样器将激光束转向90°直角棱镜（RAP1）。 </li><li>使用另一个直角棱镜（RAP2）允许光束通过可变中性密度滤光片（NDF2; OD = 0-4.0）并通过多模光纤（MMF），将其引导通过光纤耦合器（FC）目标组合（数值孔径（NA）:0.25）和XY平移器。 </li><li>使用XY转换器将光纤固定到扫描台上。将距离光纤输出端25毫米的平凸透镜（L1）放置在光纤以外的位置。 </li><li>将准直光束通过顶角为130°的锥形透镜，以产生环形光束。使用自旋式光学冷凝器（OC），锥形角度为70°和110°并在中心有一个孔，将环形光束轻微聚焦到被摄体上。 </li><li>在自制冷凝器的中心放置一个带有声透镜（AL）的50 MHz超声波换能器（UST）。 </li></ol></li><li> 系统配置:OR-PAM <ol><li>用一个由二极管泵浦的固态Nd-YAG激光器（532nm）和可调谐范围为559-576nm的染料激光器组成的纳秒可调谐激光器系统作为光学照射源。使用外部控制器将激光波长设置为570 nm，使用激光软件将激光重复率设置为5 kHz。 </li><li>将计算机控制的旋转台（保持RAP1）旋转90°，将激光束转移到光圈上进行重新整形。 </li><li>沿着光束使激光束放置一个可变的中性密度滤光片（OD:0-4.0），然后用聚光透镜（CL）对光束进行聚焦。将其穿过距离CL 75 mm的针孔（PH），进行空间过滤。 </li><li>使用由0.1 NA物镜组成的单模光纤耦合器（FC）将空间滤波光束发射到单模光纤（SMF）上，以将光束聚焦到SMF上。 </li><li>调整光纤耦合器以实现最大耦合效率。 </li><li>将光纤固定到t上他使用滑板（SP）扫描舞台。放置距离SM光纤50 mm的消色差透镜（L2），以准直激光束。 </li><li>使用运动学可控椭圆镜（M）将准直光束转向90°，以填充另一个相同的消色差透镜（L3）的后孔。使用透镜管（LT）将用于聚焦的消色差透镜放置在平移支架（TM2）上。 </li><li>将聚焦光束通过由直角棱镜（RA）和菱形棱镜（RP）组成的自制光声光束组合器，其间具有一层硅油（SO）。 注意:硅油层将作为光学透明和声反射膜。 </li><li>安装声透镜（AL），以提供菱形棱镜底部的声聚焦（焦距:〜46μm）。 </li><li>将超声波换能器的中心频率为50 MHz放置在菱形棱镜的顶部;使用环氧树脂层进行有效耦合。 </li></ol></li></OL&gt; 2.系统切换和对准<ol><li>将自制的可切换板固定（通过拧紧）到由连接到计算机的3轴控制器控制的3轴电动平台。 </li><li>使用保持架安装支架将AR和OR笼系统连接到自制板上，以便在AR和OR扫描头之间轻松切换。将扫描头滑动到成像区域的顶部。 </li><li>使用Z级将AR-OR-PAM扫描仪头部的底部淹没在充满水的丙烯酸罐（13 cm x 30 cm x 3 cm）中，进行声耦合。 </li><li>打开容器底板上直径为7厘米的成像窗口，并用聚乙烯膜密封以进行光学和声学传播。 </li><li> 使用脉冲回波放大器和示波器将超声波换能器对准。 <ol><li>在发送/接收模式下，将脉冲回波放大器的增益设置为24db。 </li><li>使用同步信号fr使用脉冲回波放大器作为触发，并使用示波器检测来自玻璃滑块（从水箱底部插入）的背散射信号。 注意:幻灯片应该粘在黑色的胶带上。 </li><li>移动Z轴以最大化脉冲回波信号的幅度（在示波器上查看）。 注意:当玻璃板对焦时，回波将具有最大幅度。 </li></ol></li><li>使用BNC电缆打开激光器并将UST连接到两个放大器，每个放大器具有24 dB固定增益。 注意:放大器的输出端连接到数据采集卡（DAQ）。 </li><li>使用位于激光器前面的光电二极管（PD）的信号作为数据采集系统的触发器。 </li><li> 在AR-PAM中，改变锥形透镜（con.L）和光学聚光器（OC）之间的距离，以使从测试对象产生的光声信号（黑带粘贴在载玻片上）的振幅最大化。通过确定最大光声（PA）信号幅度，确保光和声聚焦是共焦的。 <ol><li>注意最大PA信号的延迟;请稍后再查看数据采集软件的重点。 </li></ol></li><li>松开扫描头的螺丝，并将扫描头从AR-PAM手动切换到OR-PAM。然后拧紧螺丝。 </li><li> 在OR-PAM中，改变聚焦消色差双重（透镜管（LT）内）和光声组合器之间的距离，以最大化示波器上显示的PA信号幅度。 <ol><li>注意最大PA信号的延迟。 注意:Finetuning是确定共焦排列的必要条件。 </li></ol></li></ol>实验步骤<ol><li> 横向分辨率和成像深度量化 <ol><li>使用直径为100纳米的金纳米颗粒来确定AR a的横向分辨率d OR系统。 </li><li>用等量的水稀释0.1mL的纳米颗粒溶液。将0.1 mL稀释的溶液分布在盖板上，并将其与罐下方的聚乙烯膜接触。 </li><li>在扫描之前，确保AR-PAM和OR-PAM在数据采集软件（参见材料表）中保持对焦（步骤2.8和2.10）。 注意:通过了解步骤2.9和2.10中最大PA信号的微秒延迟乘以采样率（250 MS / s），图像将在数据采集软件中处于焦点。可以在软件中确定在数据采集期间必须省​​略的延迟，以便仅保存用于后处理的必要数据点。 </li><li>设置AR-PAM的扫描参数，然后按"扫描"按钮开始光栅扫描。 <ol><li>在数据采集软件中以"4"mm / s的扫描速度在"速度"中设置AR-PAM的扫描参数;选项卡，"脉冲重复率"选项卡中的"1"kHz，"Y扫描范围"选项卡中的"0.5"mm，"X扫描范围"选项卡中的"0.5"mm。在"dx"选项卡中将x方向的步长设置为"4"μm。 注意:从方向的扫描速度速度和脉冲重复率（在这种情况下为4,000μm/ 1,000 Hz = 4μm）自动确定y方向的步长， </li></ol></li><li>设置OR-PAM的扫描参数，然后按"扫描"按钮开始光栅扫描。 <ol><li>在"速度"选项卡中，将扫描参数设置为"2.5"mm / s的扫描速度，"脉冲重复率"选项卡中的"5"kHz，"Y扫描范围"为0.5"mm，选项卡，"X扫描范围"选项卡中为"0.5"mm。在"dx"选项卡中将x方向的步长设为"0.5"μm。 注意:从台阶的扫描速度和脉冲重复率（在这种情况下，2500μm/ 5000Hz =0.5μm）自动确定y方向的尺寸。 </li></ol></li><li>确保在扫描过程中，数据被连续捕获并存储在计算机上 注意:数据将仅在Y级的一个运动方向上捕获。 </li><li>使用存储在计算机中的多个B扫描数据使用图像处理软件检索最大幅度投影（MAP）图像（参见材料表 ）。 </li><li>使用来自扫描的单个纳米颗粒图像（多个图像中的多个图像）通过手动绘制通过纳米颗粒图像的中心区域的线来确定横向分辨率，以获得看起来像高斯曲线的点扩展函数。参见图2 。 </li><li>使用Gau适应从单个纳米颗粒图像获得的点扩散函数ssian拟合函数，并使用图像处理软件测量半高宽（FWHM）（见材料表 ）。使用它作为横向分辨率。参见图2 。 </li><li>将一块黑色胶带倾斜地放在一块切片的鸡肉组织上作为目标物体进行深度成像。将纸带放在水箱中。 注意:黑色胶带卡在具有尖锐尖端的金属板上，这有助于将胶带粘贴到纸巾上。 </li><li>将AR-PAM的扫描参数设置到数据采集软件中，然后按"扫描"按钮捕获单个B扫描图像，以确定最大成像深度。 <ol><li>在"速度"标签中将扫描参数设置为"15"mm / s，"脉冲重复率"选项卡中为"1"kHz，"Y扫描范围"选项卡中为"5"cm，"0.1" "mm"在"X扫描范围"选项卡。设定t在"dx"选项卡中，在x方向上的步长为"0.1"mm。 </li></ol></li><li>设置OR-PAM的扫描参数，然后按"扫描"按钮捕获单个B扫描图像，以确定最大成像部分。 <ol><li>将数据采集软件中的扫描参数设置为"速度"选项卡中的"15"mm / s扫描速度，"脉冲重复率"选项卡中的"5"kHz，"Y扫描范围"中的"2"选项卡，"X扫描范围"选项卡中为"0.1"mm。在"dx"选项卡中，将x方向的步长设置为"0.1"mm。 注意:由于X扫描范围和dx相同，因此只能捕获一次B扫描。时间分辨的PA信号乘以软组织中的声速（1,540m / s）将产生A线图像。在Y级的连续运动期间捕获多条A线以产生B扫描。 </li></ol></li></ol></li><li> 体内&lt;/ em&gt;成像的小鼠耳血管血管<ol><li>使用体重25克，年龄4周的雌鼠。 </li><li>使用氯胺酮（120mg / kg）和西咪嗪（16mg / kg）腹腔注射（剂量为0.1mL / 10g）麻醉动物。 </li><li>使用脱毛霜从动物耳朵上取下头发。擦拭区域清洁。用无菌眼软膏覆盖动物的眼睛，以避免任何分散的激光束落在眼睛上。 </li><li>将动物放在还有一个微型板的台上定位耳朵。 </li><li>在成像期间，用吸入的异氟烷（0.75L，1L / min氧气）维持麻醉。 </li><li>将脉搏血氧计固定到鼠标腿或尾部并监测生理状态。使用超声波凝胶使成像区域与聚乙烯膜接触。 </li><li>设置AR-PAM的扫描参数，然后按"扫描"按钮启动光栅扫描ING。 <ol><li>在"速度"选项卡中，在数据采集软件中以"15"mm / s扫描速度设置AR-PAM的扫描参数，"脉冲重复率"选项卡中的"1"kHz，" Y扫描范围"选项卡，"X扫描范围"选项卡中为"6"mm。在"dx"选项卡中将x方向的步长设置为"30"μm。 注意:从方向的扫描速度和脉冲重复率（在这种情况下，为15,000μm/ 1,000 Hz = 15μm）自动确定y方向的步长。 </li></ol></li><li>完成AR-PAM扫描后，将成像头位置从AR-PAM切换到OR-PAM（如第2节所述）。 </li><li>设置OR-PAM的扫描参数，然后按"扫描"按钮开始光栅扫描。 <ol><li>在数据采集软件中以"15"mm / s扫描速度在"速度"中设置OR-PAM的扫描参数y"选项卡，"脉冲重复率"选项卡中的"5"kHz，"Y扫描范围"选项卡中的"10"mm，"X扫描范围"选项卡中的"6"mm，设置步长在"dx"标签中的x方向为"6"μm。 注意:沿着方向的扫描速度和脉冲重复率（在这种情况下为15,000μm/ 5,000 Hz = 2μm），自动确定y方向的步长。 </li></ol></li><li>使用计算机中存储的多个B扫描数据，使用图像处理软件检索MAP图像。 </li><li>在整个成像期间观察动物。 </li></ol></li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+optical-resolution+photoacoustic+microscopy.">Near-infrared optical-resolution photoacoustic microscopy.</a> Opt Lett. 39 (17), 5192-5195 (2014).</li><li>Danielli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+photoacoustic+nanoscopy.">Label-free photoacoustic nanoscopy.</a> J Biomed Opt. 19 (8), 086006 (2014).</li><li>Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflection-mode+submicron-resolution+in+vivo+photoacoustic+microscopy.">Reflection-mode submicron-resolution in vivo photoacoustic microscopy.</a> J Biomed Opt. 17 (2), 020501 (2012).</li><li>Hu, S., Maslov, K., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+optical-resolution+photoacoustic+microscopy+with+improved+sensitivity+and+speed.">Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed.</a> Opt Lett. 36 (7), 1134-1136 (2011).</li><li>Maslov, K., Zhang, H. F., Hu, S., Wang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-resolution+photoacoustic+microscopy+for+in+vivo+imaging+of+single+capillaries.">Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries.</a> Opt Lett. 33 (9), 929-931 (2008).</li><li>Upputuri, P. K., Krishnan, M., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsphere+enabled+sub-diffraction+limited+optical+resolution+photoacoustic+microscopy:+a+simulation+study.">Microsphere enabled sub-diffraction limited optical resolution photoacoustic microscopy: a simulation study.</a> J Biomed Opt. 22, 045001 (2017).</li><li>Upputuri, P. K., Wen, Z. B., Wu, Z., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+photoacoustic+microscopy+using+photonic+nanojets:+a+simulation+study.">Super-resolution photoacoustic microscopy using photonic nanojets: a simulation study.</a> J Biomed Opt. 19 (11), 116003 (2014).</li><li>Allen, T. 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所有动物实验均按照新加坡南洋理工大学机构动物保护和使用委员会（动物协议号ARF-SBS / NIE-A0263）批准的准则和条例进行。作者对手稿没有相关的经济利益，也没有其他潜在的利益冲突披露。

总而言之，已经开发出可切换的AR和OR PAM系统，其可以在更低的成像深度处实现高分辨率成像并且在较高的成像深度处实现较低分辨率的成像。确定可切换系统的横向分辨率和成像深度。该可切换PAM系统的优点包括:（1）使用紧密光学聚焦的高分辨率成像; （2）使用声学聚焦的深层组织成像; 3）AR-PAM的暗场照明，防止强烈的PA信号出现在皮肤表面; 4）将样品保持在一个地方，而不是在不同系统之间?...

高分辨率光学成像方式，如光学相干断层扫描，共聚焦显微镜和多光子显微镜，有许多好处。然而，随着成像深度的增加，空间分辨率显着降低。这是因为软组织中光传输的漫射性质1,2 。光激发和超声检测的集成提供了一种解决方案，以克服深层组织中高分辨率光学成像的挑战。光声显微镜（PAM）是一种可以提供比其他光学成像模式更深的成像的方式。已成功应用于体内结构，功能，分子和细胞成像3,4,5,6,7,8 </sup&gt; ， 9，10，11，12，13研究结合强光学吸收对比度与超声波的高空间分辨率。 在PAM中，短的激光脉冲照射组织/样品。通过发色团（ 例如，黑色素，血红蛋白，水等 ）的光吸收导致温度升高，这又导致以声波（光声波）的形式产生压力波。产生的光声波可以由组织边界外的宽带超声换能器检测。利用弱光学和紧密的声学聚焦，可以在声分辨光声显微镜（AR-PAM） 14,15,16中实现深层组织成像。在AR-PAM，横向分辨率为45μm，成像深度高达3mm，已被证明15 。为了在声学上解析单个毛细管（约5μm），需要在&gt; 400 MHz中心频率下运行的超声波换能器。在这样高的频率下，穿透深度小于100μm。使用紧密的光学聚焦可以解决紧密的聚焦造成的问题。光学分辨率光声显微镜（OR-PAM）能够分辨单个毛细管，甚至单个细胞17 ，并且已经实现了0.5μm的横向分辨率18,19,20,21,22,23,24 。使用光子纳米喷嘴可以帮助实现超出衍射极限分辨率的分辨率n 25，26 。在OR-PAM中，由于聚焦而使穿透深度受到限制，并且它可以在生物组织23内形象高达〜1.2mm 。因此，AR-PAM可以使图像更深，但分辨率更低，OR-PAM可以以非常高的分辨率进行成像，但成像深度有限。 AR和OR-PAM系统的成像速度主要取决于激光源27的脉冲重复率。 结合AR-PAM和OR-PAM将对需要高分辨率和深度成像的应用非常有利。将这些系统结合在一起已经做了很少的努力。通常，使用两种不同的成像扫描仪进行成像，这要求样品在两个系统之间移动，因此难以进行体内成像。然而，使用AR和OR PAM的混合成像可实现具有可扩展分辨率a的成像深度。在一种方法中，使用光纤束来传送用于AR和OR PAM的光。在这种方法中，使用两个单独的激光器（对于AR为570nm的高能激光器，对于OR为532nm的低能量，高重复率激光器），使得系统不方便和昂贵28 。 OR-PAM激光波长是固定的，并且使用该组合系统不可能进行许多研究，例如氧饱和度。 AR和OR PAM之间的比较研究也是不可能的，因为AR和OR之间的激光波长不同。此外，AR-PAM使用明场照明;因此，来自皮肤表面的强光电信号限制了图像质量。因此，该系统不能用于许多生物成像应用。在另一种执行AR和OR PAM的方法中，光学和超声波聚焦被移动，这使得光焦点和超声波聚焦不对齐。因此，图像质量不是最佳的<sup class ="xref"&gt; 29。使用这种技术，AR-PAM和OR-PAM可以分别达到139μm和21μm的分辨率，使其分辨率不高。据报道另一种方法是改变光纤和准直光学器件，以便在AR和OR PAM之间进行切换，使得对准过程困难。在所有这些情况下，AR-PAM都不使用暗场照明。暗场照明的使用可以减少从皮肤表面产生强烈的光声信号。因此，使用环形照明可以进行深层组织成像，因为深色光声信号的检测灵敏度将比亮场照明的检测灵敏度高。 这项工作报告了一个可切换的AR和OR PAM（AR-OR-PAM）成像系统，能够对相同样品进行高分辨率成像和低分辨率深层组织成像，使用相同的激光和扫描仪进行两个系统EMS。 AR-OR-PAM系统的性能通过使用幻影实验确定空间分辨率和成像深度来表征。 在小鼠耳朵上进行体内血液血管成像以证明其生物成像能力。

<table border="0" cellpadding="0" cellspacing="0" width="939">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Q-switched Nd:YAG laser</td>
			<td style="width:232px;">Edgewave</td>
			<td style="width:177px;">BX80-2-L</td>
			<td style="width:227px;">Pump laser&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Credo-High Repetition Rate Dye Laser</td>
			<td>Spectra physics</td>
			<td style="width:177px;">CREDO-DYE-N</td>
			<td>Dye laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision Linear Stage</td>
			<td>Physik Instrumente</td>
			<td>PLS 85&#160;</td>
			<td>XY raster scanning stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Translation stage</td>
			<td>Physik Instrumente</td>
			<td>VT 80&#160;</td>
			<td>Confocal determine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounted Silicon photodiode</td>
			<td>Thorlabs</td>
			<td>SM05PD1A</td>
			<td>Triggering/Pulse variation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Motorized continuous Rotational stage&#160;</td>
			<td>Thorlabs</td>
			<td>CR1/M-Z7</td>
			<td>Diverting laser beam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounted Continuously Variable ND Filter</td>
			<td>Thorlabs</td>
			<td>NDC-50C-4M</td>
			<td>Intensity variable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber Patch Cable</td>
			<td>Thorlabs</td>
			<td>M29L01</td>
			<td>Multimode fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope objective</td>
			<td>Newport</td>
			<td>M-10X</td>
			<td>Objective&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XY translating mount</td>
			<td>Thorlabs</td>
			<td>CXY1</td>
			<td>Translating mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA1951</td>
			<td>Collimating lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical lens&#160;</td>
			<td>Altechna</td>
			<td>APX-2-B254</td>
			<td>Ring shape beam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Translation stage</td>
			<td>Thorlabs</td>
			<td>CT1</td>
			<td>Translating stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Optical condenser</td>
			<td style="width:232px;">Home made</td>
			<td style="width:177px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonic transducer</td>
			<td>Olympus-NDT</td>
			<td>V214-BB-RM</td>
			<td>50MHz transducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plano concave lens</td>
			<td>Thorlabs</td>
			<td>LC4573</td>
			<td>Acoustic lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pulser/Receiver</td>
			<td>Olympus-NDT</td>
			<td>5073PR</td>
			<td>Pulse echo amplifier&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mounted standard iris</td>
			<td>Thorlabs</td>
			<td>ID12/M</td>
			<td>Beam shaping</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Plano convex lens</td>
			<td>Thorlabs</td>
			<td>LA4327</td>
			<td>Condenser lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mounted precision pinhole</td>
			<td>Thorlabs</td>
			<td>P50S</td>
			<td>Spatial filtering</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Single mode fiber patch cable</td>
			<td>Thorlabs</td>
			<td>P1-460B-FC-1</td>
			<td>Single mode fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fiber coupler</td>
			<td>Newport</td>
			<td>F-91-C1</td>
			<td>Single mode coupling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Achromatic doublet lens</td>
			<td style="width:232px;">Edmund Optics</td>
			<td>32-317</td>
			<td>Achromatic doublet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Protected silver elliptical mirror</td>
			<td>Thorlabs</td>
			<td>PFE10-P01</td>
			<td>Mirror</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Right angle kinematic mirror mount</td>
			<td>Thorlabs</td>
			<td>KCB1</td>
			<td>Mirror mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Z-Axis Translation Mount</td>
			<td>Thorlabs</td>
			<td>SM1Z</td>
			<td>z translator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Lens tube</td>
			<td>Thorlabs</td>
			<td>SM05L10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV Fused Silica Right-Angle Prism</td>
			<td>Thorlabs</td>
			<td>PS615</td>
			<td>Right angle prism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Rhomboid prism</td>
			<td style="width:232px;">Edmund Optics</td>
			<td>47-214</td>
			<td>Shear wave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethylpolysiloxane</td>
			<td>Sigma Aldrich</td>
			<td>DMPS1M</td>
			<td>Silicon oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Amplifier</td>
			<td>Mini Circuits</td>
			<td>ZFL-500LN</td>
			<td>Amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">16 bit high speed digitizer</td>
			<td>Spectrum</td>
			<td>M4i.4420</td>
			<td>Data acquisition card</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Oscilloscope</td>
			<td style="width:232px;">Agilent Technologies</td>
			<td style="width:177px;">DS06014A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Mice&#160;</td>
			<td style="width:232px;">InVivos Pte.Ltd</td>
			<td style="width:177px;">ICR</td>
			<td>Animal model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Ultrasound gel&#160;</td>
			<td>Progress/parker acquasonic gel</td>
			<td>PA-GEL-CLEA-5000</td>
			<td>Acoustic coupling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Water tank</td>
			<td style="width:232px;">Home made</td>
			<td style="width:177px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Translation stage</td>
			<td style="width:232px;">Homemade</td>
			<td style="width:177px;"></td>
			<td>Switching AR-OR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Gold nanoparticles</td>
			<td>Sigma Aldrich</td>
			<td align="right">742031</td>
			<td>Lateral resolution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Sterile ocular ointment</td>
			<td style="width:232px;">Alcon</td>
			<td style="width:177px;">Duratears</td>
			<td>Animal imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">1951 USAF resolution test target</td>
			<td style="width:232px;">Edmund Optics</td>
			<td align="right" style="width:177px;">38257</td>
			<td>Confocal alignment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Data acquisition software</td>
			<td style="width:232px;">National Instrument</td>
			<td style="width:177px;">Labview</td>
			<td>Home made software using Labview</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Image Processing software</td>
			<td style="width:232px;">Mathworks</td>
			<td style="width:177px;">Matlab</td>
			<td>Home made program using Matlab</td>
		</tr>
	</tbody>
</table>

switchable acoustic and optical resolution photoacoustic microscopy for in vivo small-animal blood vasculature imaging

光声显微镜（PAM）是一种快速增长的invivo成像模式，结合光学和超声波，提供高分辨率穿透超过光学平均自由程（皮肤约1毫米）。通过将光学吸收对比度与单一模态中超声的高空间分辨率相结合，该技术可以穿透深层组织。光声显微镜系统可以具有较低的声学分辨率和探针深度或较高的光学分辨率和探针。通过单个系统实现高空间分辨率和大深度穿透是具有挑战性的。这项工作提出了一个AR-OR-PAM系统，能够在浅层深度进行高分辨率成像和体内相同样品的低分辨率深层组织成像。使用光学聚焦的1.4mm成像深度的横向分辨率为4μm，使用声学聚焦的具有7.8mm成像深度的45μm的横向分辨率成功利用组合系统进行演示。在这里， 进行体内小动物血液血管成像以证明其生物成像能力。

AR-OR-PAM系统的原理图如图1所示。在这种设置中，所有组件都集成在一起，并组装在一个光学保持架中。使用笼式系统可使AR-OR-PAM扫描头紧凑，易于组装，对齐和集成到单个扫描台上。 在图像采集期间使用成像头的二维连续光栅扫描。时间分辨的PA信号乘以声速（1,540m / s）以获得A线。在Y阶段的连续运动?...

Watch this Scientific Journal Video about Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging at JoVE.com

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

这里展示了可在体内在相同样品上进行浅层深度高分辨率成像和低分辨率深层组织成像的可切换声学分辨率（AR）和光学分辨率（OR）光声显微镜（AR-OR-PAM）系统。

可切换声光学分辨率光声显微镜<em&gt;体内</em&gt;小动物血液血管成像

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

switchable-acoustic-optical-resolution-photoacoustic-microscopy-for

Research

JoVE Journal

Bioengineering

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

11.8K 次观看.  Nanyang Technological University. 这里展示了可在体内在相同样品上进行浅层深度高分辨率成像和低分辨率深层组织成像的可切换声学分辨率（AR）和光学分辨率（OR）光声显微镜（AR-OR-PAM）系统。 

视频: Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

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 ...

科研

生物工程

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

支持的脂双层云母制备的高分辨率光学显微成像

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

高性能紧凑型光声层析成像系统<em&gt;体内</em&gt;小动物脑成像

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

手持式实时非侵入性小动物成像的临床光声成像系统

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...