我们认识到，生物工程和在密苏里大学的克里斯托弗邦德生命科学中心部的支持。我们承认，从密苏里生命科学研究委员会09-1034和国立卫生研究院R21CA139186 0赠款支持。我们也感谢在密苏里州，密苏里大学分子细胞学核心，和密苏里大学工程学院的财政支持大学生命科学学院本科生研究机会计划。

1。血液样品制备<ol><li>从第四阶段的癌症患者分成两个5毫升vacutainers绘制约10毫升的全血。 </li><li>在每分钟3000转（RPM）为10分钟的离心管。 </li><li>血样将分成三个不同层次：底层包括红细胞，中间层，也称为“ 捉鬼外套，包括白细胞和黑色素瘤细胞，并在顶层包含血浆和血小板。删除尽可能多的血浆，而不会干扰捉鬼外套。 </li><li>将250μL的Histopaque 1077两个1毫升Wintrobe管。 </li><li>使用9“巴斯德吸管，提取剩余的血浆，整个捉鬼外套，甚至红细胞的顶层，一定要收集&lt;500μL。 </li><li> Histopaque层上面的解决方案，并在15 mL锥形管为10分钟3000转离心Wintrobe管。 </li><li>血液将再次分开的，但这个时候红细胞分开的Histopaque，允许容易提取的棕黄色大衣。使用9“巴斯德吸管获得整个捉鬼外套，并放置在2 mL Eppendorf管。 </li><li>暂停在1.5毫升的PBS 2％V / V吐温20捉鬼外套。 </li></ol> 2。流室建设<ol><li>获取压克力缸，外直径20毫米，17毫米内径，和8毫米高。对方在90度角，钻透一侧气缸的3个5毫米直径的孔。剪切聚合物管件（内径1.6毫米，5毫米外径），并放入钻孔。 </li><li>横跨丙烯酸环的底部封口膜，使水的密封。封口膜形式与丙烯酸环的浅碗，将持有的丙烯酰胺溶液，将形成一个流动室。 </li><li>暂停通过管件，对面是一个直径为1.6毫米的电线对方，确保单丝通过环中可见。填写一个1.6毫米的线径和位置远离暂停丝管1毫米的油管第三件。这些导线进入油管防止丙烯酰胺。丙烯酰胺固化后，将提取的电线，留下一个圆柱的流动路径和一个地方的位置光纤聚焦到流动路径。 </li><li>加入5毫升准备丙烯酰胺[20克丙烯酰胺（Sigma公司Aldrich公司），0.7克双丙烯酰胺（Sigma公司Aldrich公司），100毫升的蒸馏水]小烧杯中。测量出0.02克过硫酸铵（Sigma公司），并把它添加到烧杯中。混合搅拌棒为3分钟。添加20μL的tetramethylethylenediamene（尔科技）的烧杯中。快速倒入混合物，丙烯酸环充填顶端。几秒钟后，该解决方案将凝胶，使流动腔模具。小心拉电线，确保不打扰的丙烯酰胺凝胶。流湛BER是现在使用的准备。 </li></ol> 3。光声血流设置<ol><li>声学夫妇一个聚偏二氟乙烯压电声换能器（的Panametrics）一个以上的流动路径直接使用超声凝胶的流动腔。 </li><li>使用刺刀奥尼尔Concelman（BNC）电缆，连接传感器输入一个RITEC宽带接收机- 640A（低通滤波器，高通滤波器：1 MHz时，输入阻抗：50欧姆，放大：35分贝）。滤波器的输出连接到示波器（泰克TDS 2024B）。触发示波器，一个光电二极管（Thorlabs）成立附近的传感器是插入到示波器使用BNC电缆。 </li><li>一个T型接头（小零件）连接到一个流室的手臂。两个注射泵（布伦特里科学）连接到连接器的其他分支。一个注射器中含有空气，而其他将包含在第一部分中编写的细胞悬液。设置流速100微升/分钟。 </li><li>直到空气形式的小口袋中提取流室的小管，并插入该插槽一个0.37数值孔径1毫米直径的芯光纤。填写，以减少接口信号和增加量，适用于流动路径的激光与水的口袋。由于数值孔径的光纤和短距离流路，激光只照射在任何特定时间蛞蝓样品，确保样品直接在前面的激光是同一样品光声信号。激光的能量应约28兆焦耳/ 厘米 2的光斑大小7 毫米 2 。 </li><li>将两个注射器泵上。当两个不混溶流体到达丁字路口，他们将分割成均匀的蛞蝓和流过去的传感器。 </li><li>与纳秒脉冲激光产生的黑色素细胞的光声波照射流动的样品。由于黑色素是一个broadb和光吸收器，任何可见的激光波长可用于。 </li><li>如果信号在示波器上时，会出现一个蛞蝓上盖传感器，蛞蝓包含黑色素瘤，应通过跟踪系统，直到下降到一个集合小瓶。 </li></ol> 4。免疫细胞化学<ol><li>为了解决这些问题，以玻片，以捕捉蛞蝓一个Cytospin 4离心机（Thermo Scientific的）。到Cytofunnels放置玻片（Shandon EZ单Cytofunnel与布朗过滤卡的Thermo Scientific）和装入Cytospin 4离心机。放入100μL1％牛血清白蛋白的PBS（Sigma公司）到每一个漏斗，然后在3分钟600转离心机帮助捕获的细胞粘到载玻片。 </li><li>离心后，添加细胞样本的Cytofunnels，然后在3分钟600转离心机。再喷乙醇固定液的幻灯片。 </li><li>两次在PBS洗10分钟的幻灯片。 </li><li&gt;来阻止非特异性结合，培育每10μL山羊血清（Sigma）和90μL的PBS在室温下1小时的混合物的幻灯片。 </li><li>孵化后，倒出与初级抗体溶液在室温下湿盒1小时的幻灯片和孵化。每张幻灯片应包含1％MART - 1抗体兔提出，CD - 45抗体在小鼠中提出的1％，10％山羊血清，89％PBS。 </li><li>孵化后，在PBS洗10分钟三次幻灯片。 </li><li>幻灯片，然后在一，二级抗体溶液，有荧光抗体绑定到的孵育。混合物含有羊抗兔抗体0.05％，0.05％的羊抗鼠抗体，0.1％山羊血清，99％PBS。幻灯片在黑暗中孵育，在室温下1小时。 </li><li>孵育后，倒出的幻灯片在PBS洗为5分钟三次。 </li><li>染色细胞核，细胞孵育0.1微克/毫升的DAPI（Invitrogen公司）1分钟。然后倒出的幻灯片，并在PBS洗。 </li><li>安装一个使用20μL每张幻灯片树脂为基础的安装媒体的样品对细胞的盖玻片。透明指甲油密封盖玻片保存幻灯片。现在的幻灯片，准备用荧光显微镜成像。 </li></ol> 5。代表性的成果： <img src="/files/ftp_upload/3559/3559fig1.jpg" alt="图1"/> 图1白血细胞和黑色素细胞的光声波形显示在这里。白血细胞，没有固有的色素沉着，产生的电子噪声平线（左）没有光声波和清单。色素性黑色素瘤细胞产生强大的光声波（右）。 <img src="/files/ftp_upload/3559/3559fig2.jpg" alt="图2"/> 图2免疫细胞化学染色后，黑色素细胞培养显示，在“基本法”的DAPI信号UE同时MART1以绿色突出显示。 <img src="/files/ftp_upload/3559/3559fig3.jpg" alt="图3"/> 图3。叠加的DAPI和MART1图像，如图2与CD45，白细胞的指标，这个数字显示几个白血细胞中的黑色素细胞。

<ol>
	<li>Pantel, K., Brakenhoff, R., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">Detection clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature Reviews Cancer. 8, 329-340 (2008).</li><li>Pantel, K., Brakenhoff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4, 448-456 (2004).</li><li>Yu, M., Stott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of Cell Biology. 192, 373-373 (2011).</li><li>Weight, R. M., Dale, P. S., Viator, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+melanoma+cells+in+human+blood+using+photoacoustic+flowmetry.">Detection of circulating melanoma cells in human blood using photoacoustic flowmetry.</a> , 106-109 (1985).</li><li>Weight, R. M., Dale, P. S., Lisle, A., Caldwell, C., Viator, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+detection+of+metastatic+melanoma+cells+in+the+human+circulatory+system.">Photoacoustic detection of metastatic melanoma cells in the human circulatory system.</a> Opt. Lett. 31, 2998-3000 (2006).</li><li>Gupta, A., Kumar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geometry+on+droplet+formation+in+the+squeezing+regime+in+a+microfluidic+T-junction.">Effect of geometry on droplet formation in the squeezing regime in a microfluidic T-junction.</a> Microfluidics and Nanofluidics. 8, 799-812 (2010).</li><li>Mocellin, S., Hoon, D., Ambrosi, A., Nitti, D., Rossi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prognostic+value+of+circulating+tumor+cells+in+patients+with+melanoma:+a+systematic+review+and+meta-analysis.">The prognostic value of circulating tumor cells in patients with melanoma: a systematic review and meta-analysis.</a> Clinical Cancer Research. 12, 4605-4605 (2006).</li><li>Cristofanilli, M., Budd, G., Ellis, M., Stopeck, A., Matera, J., Miller, M., Reuben, J., Doyle, G., Allard, W., Terstappen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells,+disease+progression,+and+survival+in+metastatic+breast+cancer.">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> New England Journal of Medicine. 351, 781-781 (2004).</li><li>Cristofanilli, M., Hayes, D., Budd, G., Ellis, M., Stopeck, A., Reuben, J., Doyle, G., Matera, J., Allard, W., Miller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+a+novel+prognostic+factor+for+newly+diagnosed+metastatic+breast+cancer.">Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer.</a> Journal of Clinical Oncology. 23, 1420-1420 (2005).</li><li>Ghossein, R., Scher, H., Gerald, W., Kelly, W., Curley, T., Amsterdam, A., Zhang, Z., Rosai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+tumor+cells+in+patients+with+localized+and+metastatic+prostatic+carcinoma:+clinical+implications.">Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications.</a> Journal of Clinical Oncology. 13, 1195-1195 (1995).</li><li>Loberg, R., Fridman, Y., Pienta, B., Keller, E., McCauley, L., Taichman, R., Pienta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+isolation+of+circulating+tumor+cells+in+urologic+cancers:+a+review.">Detection and isolation of circulating tumor cells in urologic cancers: a review.</a> Neoplasia. 6, 302-302 (2004).</li><li>Viator, J. A., Gupta, S. K., Goldschmidt, B. S., Bhattacharyya, K., Raghuraman, R., Shukla, R., Dale, P. S., Boote, E., Katti, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+gold+nanoparticle+enhanced+prostate+cancer+cells+using+photoacoustic+flowmetry+with+optical+reflectance.">Detection of gold nanoparticle enhanced prostate cancer cells using photoacoustic flowmetry with optical reflectance.</a> J. Biomed. Nanotech. 6, 187-191 (2010).</li></ol>

约翰A Viator Viator科技公司，形成商业化光声技术，为改善人类健康的公司的创始人和总裁。

检测车辆检验中心仍然是极少数因难的问题隔离罕见的肿瘤细胞临床应用的研究密集型领域。许多其他技术正在评估的CTC检测，包括RT - PCR技术，微流体细胞捕捉，免疫捕获，和其他方法8-12。然而，CMC的显示光声检测的承诺，因为它是标签，并提供了一​​种快速，准确的手段捕捉小，重量轻的吸收颗粒。 协议描述白血细胞分离效率高，还存在样品中的红血细胞的低数量。这些胭脂污染的细胞也能吸收激光灯，但在一个水平大大下降。为了确保红血细胞不干扰我们的黑色素瘤的检测系统，通过该系统推出5个健康的病人样本，没有人给一个信号，表明，目前在足够低浓度的红血细胞被忽略。 使用培养的黑色素细胞和光声流系统已被证明敏感下降到1细胞/μL（未发表数据），已进行了一系列的集中研究。这些研究下去，并很快将包括使用癌症病人样本的审判。 这光声技术也正在评估中使用，如乳腺癌和附加外源性生色前列腺癌，非色素的CTC。我们已经完成前期工作，利用黄金纳米粒子癌症cells13。这些试验表明，纳米粒子可以提供必要的光吸收产生光声波。其余的技术挑战是癌细胞的选择性附加这样的粒子。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> ABCD1234 </td><td></td></tr></tbody></table>

detection and isolation of circulating melanoma cells using photoacoustic flowmetry

循环肿瘤细胞（CTCS）是那些从宏观肿瘤分离，细胞通过血液和淋巴系统种子1,2,3继发性肿瘤蔓延。车辆检验中心是转移性疾病和他们的血液样本检测指标，可用于诊断癌症和监测病人的治疗反应。由于车辆检验中心是罕见的，对肿瘤细胞之间的正常血细胞的数十亿美元，在晚期癌症患者，他们的检测和枚举组成，是一项艰巨的任务。我们利用在大多数黑色素瘤细胞色素的存在，产生的光声，或激光诱导超声波在自定义流式细胞仪检测循环黑色素瘤细胞（CMCS ）4,5。这个过程需要使用离心和取得的白血细胞层分离全血样本。如果目前在全血，由于类似密度的白血细胞的CMC将单独。这些细胞悬浮于磷酸盐缓冲液（PBS），并引入流量计。而不是一个连续流动血细胞悬液，诱导两相流为了捕捉这些细胞作进一步研究。在两相流中，在一个微流体系统中的两个互不相溶的液体在路口和蛞蝓液体6,7交替的形式满足。 PBS暂停依次用激光照射的白血细胞和空气形成微升蛞蝓。增加了液相的表面活性剂可以形成统一蛞蝓，用户可以通过改变两相的流速不同大小的蛞蝓。空气和白血细胞的PBS鼻涕虫的蛞蝓包含没有光吸收，因此，不产生光声波。然而，甚至包含单一的CMC的白血细胞，蛞蝓吸收激光和产生高频率的声波。这些蛞蝓产生的光声波，被隔离，并收集细胞化学染色为verificATION奇美通讯。

Watch this Scientific Journal Video about Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry at JoVE.com

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

我们已经开发出一种使用激光诱导超声检测循环黑色素瘤细胞的转移性疾病的早期指标的流式细胞仪。

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 ...

detection-isolation-circulating-melanoma-cells-using-photoacoustic

Research

JoVE Journal

Bioengineering

15.7K 次观看.  University of Missouri. 我们已经开发出一种使用激光诱导超声检测循环黑色素瘤细胞的转移性疾病的早期指标的流式细胞仪。

视频: Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

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

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

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 ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

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 ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

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 ...