This work was supported by a grant from the Jan Kornelis de Cock foundation.

1，创建模具硅胶用于肿瘤模拟夹杂物<ol><li>收集所希望的形状和尺寸可以作为肿瘤模拟夹杂物， 如珠子或弹子模型的固体物品。 </li><li>彻底清洁肿瘤模型。以确保容易地移除从硅酮模具中，肿瘤模型可喷施抗粘附喷涂或覆盖有一层薄薄的凡士林或蜂蜡的。 </li><li>放置在一个单独的薄壁方（塑料）中的每个模型表面光滑。如果需要的话，注视模型到盒子的底部以保持其位置。用一个框，比肿瘤模型本身，以避免浪费硅过量稍大。 </li><li>倒入所需的有机硅组分A的量在搅拌碗，并在10加有机硅组分B:1的比例（重量）。彻底混合这两种成分。任选地，真空泵可用于从硅氧烷混合物中除去气泡。 </li><li>轻轻婆乌尔在塑料盒中硅的混合物，防止截留气泡。聚硅氧烷混合物应在45分钟进行处理，以获得最佳结果。 </li><li>让切割模具，除去肿瘤模型之前硅氧烷混合物固化为至少6小时。任选地，所述有机硅模具可切割以Z字形图案，以允许它适合背部一起干净。 3天之后，得到的聚硅氧烷的最大强度。 </li></ol> 2创建的Tris-缓冲盐溶液<ol><li>创建Tris缓冲盐水（TBS）加入6.1克（50毫摩尔）的Tris和8.8克（150毫摩尔）氯化钠800毫升去离子水的溶液。 </li><li>加入将1.0g（15毫摩尔）的NaN 3的方框血红蛋白（步骤3.3和4.4）的氧化和抑制细菌生长。注意:NaN 3的是一种严重的毒。这可能是致命的皮肤或吞食接触。该化合物的毒性与可溶性碱金属氰化物和致死剂量的对于成人是约0.7g。始终遵循安全指示由制造商提供。 </li><li>将pH调节到7.4，并把该卷到1000毫升去离子水。 </li></ol> 3，建立荧光包裹<ol><li>添加2克琼脂糖至50ml TBS从步骤2琼脂糖的较高熔点相比明胶（步骤4.2）将防止夹杂物的溶解和当放置在熔化明胶漏出荧光染料。任选地，加入的琼脂糖的量可以被改变为1或3克，得到较软的或可触知的肿瘤的夹杂物，分别。 </li><li>加热使用微波，直到沸点达到琼脂糖​​浆。彻底搅拌直到琼脂糖完全溶解。 </li><li>添加1.1克（17微摩尔）的血红蛋白和5ml脂肪乳20％溶解在50毫升的TBS的琼脂糖混合物在不断搅拌下，以类似的周围乳房假体组织（步骤4）的光学特性。 </li><li>加入20.0毫克（250.8微摩尔的荧光色素吲哚花青绿83.8毫升去离子水）。确保染料完全溶解。 </li><li>吸管5.0毫升从该溶液中，并把它添加到琼脂糖混合物以获得14微米的最终浓度。任选地，可以使用其他荧光染料相比ICG如果需要的话用自己的最佳浓度。 </li><li>轻轻地填充在步骤1与使用注射器（ 图1A）的热琼脂糖混合物中创建的硅氧烷模具。重复这个过程，直到所有的模具都充满。 </li><li>让荧光夹杂物固化在室温约1小时。由覆盖有铝箔的整个模具保护从光的夹杂物。 </li><li>凝固后，轻轻地打开模具和压出的夹杂物（ 图1B）。或者，使用注射器的针尖对夹杂物的表面上应用小水珠融化的琼脂糖混合物。通过重复此过程数次的同一位置上，小督铁道部马刺可以创建模拟浸润性肿瘤。 </li><li>避光和脱水琼脂糖夹杂物通过它们包装在铝箔和它们在潮湿的储存容器储存于4℃。 注:较低或较高的荧光染料浓度比使用已知浓度的优化都将会导致减少的荧光信号强度。看似有悖常理降低信号强度随上述最佳荧光染料浓度的染料浓度是由于这种现象称为淬火。当评估在幻影的荧光染料的最大深度渗透，用最适浓度是必要的。 </li></ol> 4，创建乳房幻影<ol><li>获取一个杯形的模具制造所需的尺寸和体积， 如玻璃或塑料碗的乳房假体。模具应具有平滑的表面，以防止凝胶的形式附着于模具。的模具VOLUM电子500毫升将创建一个具有相当规模的乳房假体。 </li><li>要创建一个乳房假体与500毫升的体积，加入50克明胶250开花至500ml的TBS（步骤2）。加热在不断搅拌下将明胶浆液至50℃。 </li><li>一旦明胶完全溶解后，让明胶混合物逐渐冷却并在35℃的恒定温度下用热水浴维持它。 </li><li>在恒定搅拌下，加入5.5克（85毫摩尔），牛血红蛋白和25ml脂肪乳20％，分别模拟吸收的光子和散射在组织。 </li><li>预冷的杯形模具中在4℃下至少1小时。接着，倒入模具中的凝胶混合物以对应于琼脂糖肿瘤模拟夹杂物（ 图1C）的预定深度的水平。让明胶混合物固化在4℃下进行30分钟至1小时。 </li><li>凝固后，放置一个肿瘤模拟荧光琼脂糖夹杂物的表面上<html>幻影暂时注视列入用小针。最多3肿瘤模拟荧光灯的夹杂物可以在一个单一的乳房假体被引入。足够的空间（至少5厘米）应保持个人肿瘤模拟夹杂物（ 图1D）之间。 </li><li>在剩下的模具量倒入温热的明胶混合的其余部分，允许两层的坚持而不产生折射文物。标记在模具上的荧光肿瘤模拟夹杂物的位置。让幻影凝固在o / n 4℃。 </li><li>一旦固化，取出用于夹杂物临时固定的针头，轻轻将其从模具中（ 图1E）的乳房假体。通过将其包装在铝箔避光和脱水的乳房假体，它在湿润的储存容器储存于4℃。 </li></ol><html>茜1"FO:内容宽度="5英寸"SRC ="/文件/ ftp_upload / 51776 / 51776fig1highres.jpg"WIDTH ="500"/&gt; 图1的序列的步骤创建一个包含荧光肿瘤模拟夹杂乳房假体的。创建所需的形状和尺寸的模具硅胶后，将模具填充有使用注射器（A）的熔化琼脂糖混合物。不同大小和形状的肿瘤模拟夹杂物产生的在目前的研究（B）。接着，薄薄的一层熔胶的混合物倒入一个定制的涂木乳腺模具（C）。凝固后，将肿瘤模拟夹杂物被定位时，暂时固着，并覆盖着熔化明胶混合物（D）的另一个层。凝固后，乳房假体是轻轻地从模具中（E）中删除。幻像然后可以应用用于模拟各种NIRF成像应用（F）。REF ="/文件/ ftp_upload / 51776 / 51776fig1highres.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 5，设置NIRF摄像系统<ol><li>需要术中应用程序中的NIRF摄像系统，模拟针对性NIRF成像在乳腺癌手术。目前可用于研究几个NIRF成像系统进行实时术NIRF成像。虽然这些设备之间的一些差异存在，它们都包含一个激励光源（用于荧光肿瘤夹杂物的激发）和用于检测发射的光子的高度敏感的成像装置。 </li><li>请务必使用足够的波长的激发光源。对于含有ICG肿瘤模拟夹杂物，使用的激励光源（ 如激光），其发射750和800纳米之间的光子。如果一个备选的荧光染料的情况下，激发波长应调整符合的毫安<html>生产商产生的说明。 </li><li>若NIRF相机系统包含一个发射滤波器滤除不需要的背景信号，确保正确的过滤器使用。对于含有ICG肿瘤模拟夹杂物，用800和850纳米之间的发射滤波器。替代性的荧光染料，可能需要不同的发射器，根据不同的制造商的说明进行操作。 注意:请确保有激发和发射波长，以防止过饱和的图像之间的零重叠。此外，该图像捕获时间可能要进行调整，以获得最佳的荧光图像。在深层次的荧光夹杂物或弱荧光信号的情况下，图像采集时间可以增加多达几秒钟到最小。在浅的夹杂物或强的荧光信号的情况下，采集时间可以降低到几毫秒，以允许在实时视频速率荧光成像。 </li></ol>E"&gt; 6。仿真技术在乳腺癌手术NIRF成像中的应用<ol><li>以从容器中组织模拟乳房假体，并将其放置在平坦的非荧光面。接着，放置上述的乳房假体的NIRF成像装置中，留下了足够的工作距离为肿瘤模拟夹杂物的切除。 </li><li>利用本地化NIRF成像和/或幻象乳房触诊肿瘤模拟荧光包容。万一没有荧光信号可被检测，则夹杂物或者定位在假想的检测或图像采集时间应增加太深。 </li><li>一旦列入本地化​​，切开假体丰胸和消除肿瘤，包括模拟实时NIRF指导下，采用传统的手术器械。可选地，包含可切下单独引导通过目视检查和乳房假体的触诊来模拟的护理标准。 </li><li>拆除后直接的肿瘤模拟夹杂物，像术腔的任何剩余的荧光活性指示切除不充分的。 </li><li>在任何情况下，剩余的荧光活动，消费NIRF直接指导下，纳入剩余直到没有荧光信号离开了。 </li><li>形象切除假体片段，模拟NIRF引导宏观边缘状态的评估。至此，切片3幽灵组织 - 5毫米斑和图像的斑块相应。荧光信号到达入手术切缘表示手术切缘阳性的存在。 </li></ol>

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我们通过使用乳房形幻影集成肿瘤模拟夹杂模拟NIRF制导BCS的潜在的临床应用。术中肿瘤的定位，NIRF引导下肿瘤切除术，手术的程度的评估，以及手术切缘术后评估中使用自定义构建NIRF摄像系统中的所有可行的。非侵入性的检测荧光肿瘤模拟夹杂物仅是为夹杂物定位在虚线组织在2厘米或更小的深度是可行的。术，但是，在有限的信号的穿透深度在很大程度上解决了由BCS手术的性质，其中，所述外...

保乳手术（BCS），其次是放射治疗是乳腺癌患者的标准治疗为T 1-T 2乳腺癌1,2。不准确的手术结果的阳性手术切缘在20至40％的谁接受BCS，因此需要附加的外科手术或放射治疗3,4,5病人的程度术评估。虽然广泛切除邻近健康乳房组织可能会降低手术切缘阳性的频率，这也将阻碍美容效果，并增加合并症6,7。因此，需要新的技术，提供了对原发肿瘤的位置和手术的程度术的反馈。光学成像，特别是近红外荧光（NIRF）成像，可以通过提供的外科医生用的r中前和术中肿瘤的定位工具减少以下BCS阳性手术切缘的频率EAL时间。最近，我们小组汇报了卵巢癌患者先在人类试验的肿瘤靶向荧光成像，显示出这项技术来检测原发肿瘤及腹腔转移癌具有较高的敏感性8的可行性。在继续到临床研究中的乳腺癌患者，但是，各种肿瘤靶向NIRF成像应用中的BCS的可行性已经可以使用临床前体模进行评价。 下面的研究方案介绍含有荧光肿瘤模拟夹杂9组织模拟乳房假体的使用NIRF成像。幽灵提供了一个廉价和灵活的工具来模拟前和术中肿瘤定位，实时NIRF引导肿瘤切除术，对手术切缘状况进行评估，并检测残留病灶。凝胶状幻影具有类似于人体组织的弹性属性，并且可以使用常规s中被切urgical仪器。在模拟的手术过程中，外科医生通过触觉信息（在可触知的夹杂物的情况下）和手术区域的视觉检查指导。此外，NIRF成像应用，以提供对手术的程度实时术中反馈的外科医生。 但是应当强调的是，NIRF成像需要使用的荧光染料。理想地，荧光染料，应使用能发出在近红外光谱范围内的光子（650 - 900纳米），以尽量减少由分子在组织生理学上丰富的吸收的光子和散射（ 例如 ，血红蛋白，脂质，弹性蛋白，胶原，和水） 10,11。而且，自体荧光（ 即，在由于在活细胞中的生化反应组织中的固有荧光活性）被最小化，在近红外光谱范围内，产生最佳的肿瘤-背景比11。由共轭NIRF染料肿瘤精华素泰德部分（ 例如，单克隆抗体），可用于术中成像应用而获得的靶向递送的荧光染料。 由于人的眼睛是不敏感的，在近红外光谱范围内点亮，高灵敏度的摄像机装置所需的NIRF成像。已经开发了几个NIRF成像系统，术中使用迄今12。在当前的研究中，我们使用一个自定义的构建是在术中的合作与应用慕尼黑工业大学开发NIRF成像系统。该系统允许同时获取彩色图像和荧光图像。以提高荧光图像的精度，校正方案为光强变化的组织实施。详细描述通过Themelis 等人提供的。13

<table><tbody><tr><td>Bovine hemoglobin</td><td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td><td>H2500</td><td>Simulates absorption of photons in tissue&amp;nbsp;</td></tr><tr><td>Intralipid 20%</td><td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td><td>I141</td><td>Simulates scattering of photons in tissue</td></tr><tr><td>Silicone A translucent 40 (2-components poly-addition silicone)</td><td>NedForm, Geleen, The Netherlands</td><td></td><td>Package consists of components A and B, that should be mixed one on one (A:B=10:1).&amp;nbsp; Link to manufacturers page: http://tinyurl.com/ncjq7jx</td></tr><tr><td>Gelatine 250 Bloom</td><td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td><td>48724</td><td>Construction of breast-shaped phantoms</td></tr><tr><td>Agarose</td><td>Hispanagar, Burgos, Spain</td><td></td><td>Construction of tumor-simulating inclusions</td></tr><tr><td>Tris</td><td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td><td>T1503&amp;nbsp;</td><td></td></tr><tr><td>HCl</td><td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td><td>258148</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td><td>S9888</td><td></td></tr><tr><td>NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck, Darmstadt, Germany</td><td>822335</td><td>CAUTION: severe poison. The toxicity of this compound is comparable to that of soluble alkali cyanides and the lethal dose for an adult human is about 0.7 grams.</td></tr><tr><td>Examples of NIRF imaging devices for intraoperative application:</td><td></td><td></td><td></td></tr><tr><td>T2 NIRF imaging platform&amp;nbsp;</td><td>SurgVision BV, Heerenveen, The Netherlands</td><td></td><td>Customized NIRF imaging system used in the current study. More details available at www.surgvision.com</td></tr><tr><td>Photodynamic Eye</td><td>Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany</td><td>PC6100</td><td>www.iht-ltd.com</td></tr><tr><td>FLARE imaging system kit</td><td>The FLARE Foundation Inc, Wayland, MA, USA</td><td></td><td>www.theflarefoundation.org</td></tr><tr><td>Fluobeam</td><td>Fluoptics, Grenoble, France</td><td></td><td>www.fluoptics.com</td></tr><tr><td>Artemis handheld camera</td><td>Quest Medical Imaging BV, Middenmeer, the Netherlands</td><td></td><td>www.quest-mi.com</td></tr><tr><td>Examples of NIRF fluorescent dyes for intraoperative application:</td><td></td><td></td><td></td></tr><tr><td>Indocyanine green</td><td>ICG-PULSION,&amp;nbsp; Feldkirchen, Germany</td><td>PICG0025DE&amp;nbsp;&amp;nbsp;</td><td>Clinical grade fluorescent dye for NIRF imaging used in the current study. More details available at www.pulsion.com</td></tr><tr><td>IRDye 800CW NHS Ester</td><td>LI-COR Biosciences, Lincoln, NE, USA</td><td>929-70021</td><td>www.licor.com</td></tr></tbody></table>

tissue-simulating phantoms for assessing potential near-infrared fluorescence imaging applications in breast cancer surgery

不准确的术中肿瘤的定位及手术切缘状态结果在保乳手术（BCS）的次优结果的评价。光学成像，特别是近红外荧光（NIRF）成像，可以通过提供的外科医生用在实时前和术中肿瘤的定位工具降低阳性手术切缘的下面BCS的频率。在目前的研究中，NIRF引导BCS的潜在利用组织模拟乳房假体的标准化和培训的原因进行了评价。 乳房假体与媲美正常乳腺组织的光学特性来模拟乳腺癌保乳手术。肿瘤模拟含有荧光染料吲哚菁绿（ICG）的夹杂物被结合在体模在预定的位置和成像前和术中肿瘤的定位，实时NIRF引导肿瘤切除术，NIRF制导评估手术的程度，以及手术切缘术后评估。定制的NIRF相机作为一种临床原型成像的目的。 含肿瘤模拟夹杂物的乳房假体提供了一个简单，价格低廉，用途广泛的工具来模拟并评估术中肿瘤成像。凝胶状幻影具有类似于人体组织的弹性属性，并且可以使用常规的外科手术器械进行切割。此外，该模体含有血红蛋白和脂肪乳用于模仿吸收的光子和散射，分别建立类似于人类乳腺组织均匀的光学特性。 NIRF成像的主要缺点是有限的穿透深度的光子穿过组织，从而阻碍深层肿瘤（非侵袭性）成像反射照明策略传播时。

从本研究结果已经预先别处9报告。 我们的数据表明，NIRF成像可应用于检测荧光肿瘤模拟夹杂物在组织模拟乳房假体，模拟NIRF制导保乳手术的乳腺癌患者。使用我们的幻象模型，我们发现术中肿瘤的定位，NIRF引导肿瘤切除术，手术腔边缘的术评估，并检测残留病是可行的（图2）。简单地说，一共有4乳房假体的制作，都包含两个荧光夹杂有明显的尺寸和/...

Watch this Scientific Journal Video about Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery at JoVE.com

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

组织鬼怪模拟，以评估乳癌手术潜在的近红外荧光成像中的应用

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

tissue-simulating-phantoms-for-assessing-potential-near-infrared

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.1K 次观看.  University Medical Center Groningen. Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes. 

视频: Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

科研

癌症研究

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

幻影使用肿瘤尺寸测量的质量保证组织模仿材料的潜伏期多模工程

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

生物工程

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

电脑断层导引的时域漫荧光体层摄影术在小动物的肿瘤生物标志物的本土化

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

医学

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Multimodal 3D Printing of Phantoms to Simulate Biological Tissue

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of an optical imaging device are greatly affected by the performance characteristics of its components, the test environment, and the operations. Therefore, it is necessary to calibrate these devices by traceable phantom standards. However, most of the currently available phantoms are homogeneous phantoms that cannot simulate multimodal and dynamic characteristics of biological tissue. Here, we show the fabrication of heterogeneous tissue-simulating phantoms using a production line integrating a spin coating module, a polyjet module, a fused deposition modeling (FDM) module, and an automatic control framework. The structural information and the optical parameters of a &#34;digital optical phantom&#34; are defined in a prototype file, imported to the production line, and fabricated layer-by-layer with sequential switch between different printing modalities. Technical capability of such a production line is exemplified by the automatic printing of skin-simulating phantoms that comprise the epidermis, dermis, subcutaneous tissue, and an embedded tumor.

Spin coating, polyjet printing, and fused deposition modeling are integrated to produce multilayered heterogeneous phantoms that simulate structural and functional properties of biological tissue.

幻影的多模态3D打印模拟生物组织

Biomedical optical imaging is playing an important role in diagnosis and treatment of various diseases. However, the accuracy and the reproducibility of ...

工程学

Terahertz Imaging and Characterization Protocol for Freshly Excised Breast Cancer Tumors

This manuscript presents a protocol to handle, characterize, and image freshly excised human breast tumors using pulsed terahertz imaging and spectroscopy techniques. The protocol involves terahertz transmission mode at normal incidence and terahertz reflection mode at an oblique angle of 30&#176;. The collected experimental data represent time domain pulses of the electric field. The terahertz electric field signal transmitted through a fixed point on the excised tissue is processed, through an analytical model, to extract the refractive index and absorption coefficient of the tissue. Utilizing a stepper motor scanner, the terahertz emitted pulse is reflected from each pixel on the tumor providing a planar image of different tissue regions. The image can be presented in time or frequency domain. Furthermore, the extracted data of the refractive index and absorption coefficient at each pixel are utilized to provide a tomographic terahertz image of the tumor. The protocol demonstrates clear differentiation between cancerous and healthy tissues. On the other hand, not adhering to the protocol can result in noisy or inaccurate images due to the presence of air bubbles and fluid remains on the tumor surface. The protocol provides a method for surgical margins assessment of breast tumors.

Freshly excised human breast cancer tumors are characterized with terahertz spectroscopy and imaging following fresh tissue handling protocols. Tissue positioning is taken into consideration to enable effective characterization while providing analysis in a timely manner for future intraoperative applications.

新切除乳腺癌肿瘤的Terahertz成像和表征协议

This manuscript presents a protocol to handle, characterize, and image freshly excised human breast tumors using pulsed terahertz imaging and spectroscopy ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

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

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

含有宏观结构的光学组织幻影的制备与表征

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

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

基于琼脂糖组织模拟光学幽灵的漫反射光谱研究

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

患者特异性聚乙烯酒精幻影制造与超声波和X射线对比脑肿瘤手术规划

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.

The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.

用于血管和肿瘤成像的明亮NIR-II荧光探针

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing ...