作者欣赏Lynnea Brumbaugh博士的仔细阅读的手稿。这项工作是由国家卫生赠款R01 EB000712，R01 EB008085，R01 CA134539，U54 CA136398和5P60 DK02057933研究院赞助。丽红五，王教授已在Microphotoacoustics，公司和恩德拉号，公司，金融利益，但不支持这项工作。

1。系统配置<ol><li>光照射<ol><li>光照射源：二极管泵浦固态激光脉冲（INNOSLAB，Edgewave）和一个染料激光器（CBR - D Sirah）。 </li><li>输出的激光束（脉冲宽度：7 NS）的重点是通过聚光镜（LA1131，Thorlabs）传递通过一个50微米的小孔（P50C，Thorlabs）。 </li><li>针孔的位置，从稍远的聚光镜的焦点相匹配的有效的空间滤波的基本模式的光束直径的针孔的直径。 </li><li>过滤后的光束衰减是一个中性密度过滤器（NDC - 50C - 2M，Thorlabs）和耦合到单模光纤（P1 - 460A - FC - 2，Thorlabs）。 </li><li>光纤输出填补显微镜的目标回光圈（RMS4X，Thorlabs）达到衍射极限〜2.6微米的光集中在570 nm波长。 </li></ol></li><li>超声检测<ol><li>超声换能器：50 - MHz的中心频率（V214 - BB - RM，奥林巴斯NDT）。 </li><li>超声换能器，超声波检测，这是符合衍射极限的光学照射同轴连接到自制的声光光束合成 11 。 </li><li>一个球形空腔地面合成底部产生声学透镜。这种声学透镜在水中的数值孔径为0.5，并在50 MHz的中心频率，给出了一个声学直径43微米的焦点。 </li><li> confocally，以最大限度地提高检测灵敏度的光学和声学重点​​是一致的。 </li></ol></li><li>声学耦合<ol><li>干式超声耦合是避免淹没在水中的动物实验，这是在早期的光声成像系统12。 </li><li>成像窗口中打开培养皿（直径9厘米）的底部，并用超声波和光学透明的聚乙烯膜密封。 </li><li> （图像清晰，SonoTech）之间的聚乙烯膜和对象所产生的光声波从对象到培养皿中，和去离子水的培养皿中进一步夫妇波的淹没或PAM的成像头成像夫妇的超声波凝胶。 </li></ol></li><li>电子<ol><li>超声换能器检测到的光声信号放大由两个级联的放大器（ZFL 500LN，Mini - Circuits公司） </li><li>放大后的信号是数字化的14位数据采集（DAQ）板在200 MS / s的采样率（CompuScope 14200，量具应用科学） </li></ol></li><li>扫描计划<ol><li>二维（2 - D）OR - PAM的成像头沿水平（XY）平面光栅扫描控制由一台个人电脑，这将触发DAQ板卡和泵浦激光器。从DAQ板的时钟信号同步的触发信号。 </li><li>快轴的2 - D扫描仪定义为横断面扫描（B扫描）的方向。 </li><li> B -扫描图像序列可以获取翻译沿慢轴成像头，形成一个体积的形象，这可以被视为直接的3 - D渲染，或在一个2 - D最大振幅预测（MAP）图像。 </li></ol></li></ol> 2。系统校准<ol><li>使用脉冲回波超声和超声波的反射来确定声焦平面的位置（即最大的超声波脉冲回波信号触发信号的时间延迟）。这一步是必需的，要建设或PAM系统时，只执行一次。 </li><li>单模光纤的耦合效率最大化。 </li><li>应用超声波凝胶的光学吸收的对象（例如，一块黑胶带）上，并轻轻重视下成像窗口充满去离子水的培养皿中。 </li><li>下入水成像头，并删除声学透镜下的气泡。 </li><li>调整成像头，直到吸收对象的光声信号从声焦平面，可以从声波延迟判断。 </li><li>调整显微镜物镜的垂直位置（即Z位置），以最大限度地从平面物体产生的光声信号的振幅。最大化的信号幅度表明，光的重点是在垂直方向的声重点相一致。 </li><li>调整显微镜物镜的水平位置（即x和y位置），直到从目标产生的光声信号，显示了对称格局。对称性表明，光的重点是在水平方向的声重点相一致。 </li><li>重复步骤2.6和2.7，直到光声信号是对称和幅度都进行了优化。 </li></ol> 3。阿sa<html> mple实验过程在体内或- PAM的小鼠耳廓血管<ol><li> 这一步是不需要裸鼠 。麻醉动物腹腔注射鸡尾酒[配方：氯胺酮（100毫克/毫升）1毫升，0.1毫升甲苯噻嗪（100毫克/毫升）和8.9 ml生理盐水;用量：0.1 ml/10 G]。剃须在耳后的头发，并进一步脱毛Surgi霜（＃：82565类别，美国国际实业）用去离子水清洗前残留的头发。请注意，如脱毛可能会略有刺激皮肤血管，因而是最好的执行计划实验前24小时。 </li><li>打开光声激光系统，并检查系统对齐。 </li><li>麻醉吸入气体（典型的流量是1.0-1.5升/分钟，根据动物的体重）3％异氟醚蒸发鼠标，保持整个实验过程中，用1％异氟醚麻醉。医疗级空气吸入气体维持在正常生理状态的鼠标建议。 </li><li>鼠标转移到一个立体阶段，并控制其体温在37 ° C加热垫。 </li><li>小鼠耳廓上的塑料板拼合，适用于耳上的超声凝胶层。避免被困里面的凝胶气泡。然后，将耳下成像窗口和超声凝胶接触的聚乙烯膜的底部慢慢抬高，直到动物阶段。软接触是必要的，因为膜贴着耳朵，可能会影响耳血流量。 </li><li>钳脉搏血氧仪，鼠标的腿或尾巴，监测其生理状态，适用于软膏的眼睛，以防止干燥和意外激光鼠标眼睛的损害。 </li><li>下声学透镜成像头，直到去离子水浸泡，并删除声学透镜下的气泡。 </li><li>激光能量密度检查，以确保它在激光安全标准是美国国家标准学会13。激光能量密度不应超过20兆焦耳/厘米2，到80时重点是皮肤表面的下方，在150微米的激光束的激光脉冲能量NJ 。 </li><li>设置激光外部触发模式，并开始试扫描。直到最强的光声信号从声焦平面成像头的Z位置调整。 </li><li>正确的扫描参数设置，并开始正式的图像采集。 </li><li>实验后，关闭激光，解除成像头去离子水，降低动物的阶段，释放鼠标，清洁与去离子水的小鼠耳，关闭麻醉系统和温度控制器，和卸载鼠标从立体定向阶段。 </li><li>如果需要重复成像，孵化器在鼠标与环境温度在37 ° C。返回鼠标的动物设施后自然醒来。否则，按照协议euthanatize和处置动物。 </li></ol> 4。功能或PAM的总浓度和血红蛋白的氧饱和度<ol><li>氧合血红蛋白（HBO 2） ​​和脱氧血红蛋白（HBR）是血红蛋白的两种主要形式，在可见光谱范围内的主要内源性的光声源。 HBO 2和HBR有不同的吸收光谱，从而可以光谱区别量化的总浓度（HBT）和血氧饱和度（SO 2）血红蛋白5。这里有两个准则，以帮助选择适当的光波长，所以测量： </li><li>波长应选择内的血红蛋白的吸收光谱（即550-600纳米）的Q带，以确保足够的信号噪声比和充分的渗透。 </li><li> HBO 2和HBr的吸收系数有明显的差异（例如，HBR主导的561 nm和HBO 2主导的578纳米）的波长建议。 </li></ol>除了​​SO 2，HBT可以计算加入HBR]和[HBO 2]在一起，或者也可以直接测量血红蛋白的摩尔消光系数的光谱（例如，530纳米，545纳米，570纳米，和的isosbestic波长584纳米）14，其中2 HBR和HBO有平等的摩尔消光系数。 5。代表性的成果： 在图1所示的血管解剖和SO 2裸鼠生活在一个由双波长（561和570 nm处）或- PAM的成像耳朵。典型的双波长图像采集时间，所以这样一个感兴趣的区域进行测量（图像尺寸：10毫米× 10毫米;步长：2.5微米× 5微米）〜 80分钟。 p_upload/2729/2729fig1.jpg“ALT =”图1“/&gt; 图1 在体内的光学分辨率的光声显微镜。 （一）总血红蛋白浓度显示血管解剖（在570 nm处获得）和（二）血红蛋白氧饱和度（在561 nm和570 nm处获得）在裸小鼠耳的地图图像。 （三）在面板的盒装地区（一）特写。在面板的规模巴（a）适用于上述（a）及（B）。

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	<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, 929-931 (2008).</li><li>Zhang, C., 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=Subwavelength-resolution+label-free+photoacoustic+microscopy+of+optical+absorption+in+vivo.">Subwavelength-resolution label-free photoacoustic microscopy of optical absorption in vivo.</a> Opt. Lett. 35, 3195-3197 (2010).</li><li>Wang, Y., Maslov, K., Kim, C., Hu, S., Wang, L. 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符合所有实验动物的程序进行，由学校在圣路易斯华盛顿大学医学院动物研究委员会批准的协议实验室动物。

在这段视频中，我们提供了一个OR - PAM的实验方案，包括系统配置，系统校准，典型的实验程序的详细说明。无标签，无创或PAM，使微血管功能和单一的毛细血管基础代谢的研究，从而有潜力，扩大我们的微循环相关的生理和病理的认识。 Microphotoacoustics是目前制造这样或PAM系统。

自制的声光光束合成： <ul><li>直角棱镜（NT32 - 545，埃德蒙光学） </li><li>菱形棱镜（NT49 - 419，埃德蒙光学） </li><li>硅油（1000cSt，Clearco产品） </li><li>或PAM系统（Microphotoacoustics） </li></ul>

three-dimensional optical-resolution photoacoustic microscopy

光学显微镜，在细胞和细胞器水平提供有价值的见解，已被广泛认可，作为一个有利的生物医学技术。由于三维（3 - D） 在体内的光学显微镜，single-/multi-photon荧光显微镜和光学相干断层扫描（OCT）已证明其非凡的灵敏度荧光和光散射对比，分别支柱。然而，生物组织，其中重要的生理/病理信息进行编码，的光吸收对比尚未被课税。 生物医学photoacoustics的出现，导致了光学显微镜的光学分辨率的光声显微镜（或- PAM），1光的照射下，其中的重点是实现蜂窝1或2级甚至亚细胞横向分辨率的衍射极限的一个新的分支。作为一个有价值的补充，现有的光学显微镜技术，OR - PAM的带来了至少两个新奇。首先和最重要的是，OR - PAM的检测光吸收对比，具有非凡的灵敏度（即100％）。 3荧光显微镜或光散射基于10月4日 （或同时）或- PAM的结合，提供全面的生物组织的光学性质。第二，OR - PAM编码成声波的光吸收，相反，在荧光显微镜和华侨城纯光学过程，并提供背景，免费检测。或PAM的声波探测减轻光散射信号衰减的影响和自然消除可能干扰之间的激发和检测，这是由于激发光谱和荧光光谱之间的重叠，在荧光显微镜常见的问题（即串扰）。 OR - PAM的独特的光吸收成像，已经证明其发明以来广泛的生物医学应用，包括但不限于，神经病学5，6，7眼科，8，9血管生物学和皮肤病10。在这段视频中，我们教系统的OR ​​- PAM的配置和调整，以及在体内功能微血管成像实验程序。

Watch this Scientific Journal Video about Three-dimensional Optical-resolution Photoacoustic Microscopy at JoVE.com

Three-dimensional Optical-resolution Photoacoustic Microscopy

光学分辨率的光声显微镜（或PAM），是一种新兴技术，成像光吸收能力对比<em&gt;在体内</em&gt;细胞的分辨率和灵敏度。在这里，我们提供了一个典型的可视化指令OR - PAM的实验方案，包括系统配置，系统校准，<em&gt;在体内</em&gt;实验程序和功能成像计划。

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 ...

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17.7K 次观看.  Washington University in St. Louis. 光学分辨率的光声显微镜（或PAM），是一种新兴技术，成像光吸收能力对比在体内细胞的分辨率和灵敏度。在这里，我们提供了一个典型的可视化指令OR - PAM的实验方案，包括系统配置，系统校准，在体内实验程序和功能成像计划。

视频: Three-dimensional Optical-resolution Photoacoustic Microscopy

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

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 ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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, ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

三维纸基微流体装置的制造用于免疫分析

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

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

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.