新切除乳腺癌肿瘤的Terahertz成像和表征协议
Summary
新鲜切除的人类乳腺癌肿瘤的特点是使用太赫兹光谱和成像遵循新鲜的组织处理协议。组织定位被考虑在内，以实现有效的表征，同时及时为将来的术中应用提供分析。
Abstract
本手稿提供了一个协议，使用脉冲太赫兹成像和光谱技术处理、描述和成像新切除的人类乳腺肿瘤。该协议涉及在正常入射处的太赫兹传输模式和斜角为30°的太赫兹反射模式。收集的实验数据表示电场的时域脉冲。通过切除组织上的固定点传输的太赫兹电场信号通过分析模型进行处理，以提取组织的折射率和吸收系数。利用步进电机扫描仪，从肿瘤的每个像素反射出太赫兹发射脉冲，提供不同组织区域的平面图像。图像可以在时间或频域中显示。此外，利用每个像素的折射率和吸收系数的提取数据，提供肿瘤的图谱太赫兹图像。该协议表明癌组织和健康组织之间的明显区别。另一方面，由于肿瘤表面存在气泡和液体，不遵守协议可能会导致图像发出嘈杂或不准确。该协议提供了一种乳腺肿瘤的手术边缘评估方法。
Introduction
在过去的十年里，Terahertz （THz） 成像和光谱学一直是一个快速增长的研究领域。在 0.1-4 THz 范围内持续开发更高效、更一致的 THz 发射器，使得其应用显著增长1。THz 已显示出希望和显著增长的一个领域是生物医学领域2。THz辐射已被证明是非电离和生物安全在功率水平通常用于分析固定组织3。因此，THz成像和光谱学被用来分类和区分各种组织特征，如含水量，以指示烧伤损伤和愈合4，肝硬化5，和癌症在切除组织6,6，7。癌症评估特别涵盖广泛的潜在的临床和外科应用，并已调查脑8，肝脏9，卵巢10，胃肠道11，和乳房7，12，13，14，15，16，17，18，1912,13,14,15,16,17,18的19癌症。7
THz乳腺癌的应用主要侧重于通过保证金评估支持乳房保护手术或乳房切除术。乳房切除术的目的是切除肿瘤和周围健康组织的一小层，与切除整个乳房的全乳房切除术形成鲜明对比。切除组织的手术边缘通过病理学进行评估，一旦样品固定在正式内，切片，嵌入石蜡，并安装在显微镜幻灯片上的4 μm×5μm切片。这个过程可能非常耗时，如果观察到正差值20，则需要稍后进行二次外科手术。美国放射肿瘤学会目前的指导方针将这种正差定义为癌细胞接触表面边缘墨水21。高吸收水合组织的THz成像主要限于表面成像，根据组织类型进行一些不同的穿透，足以满足快速边缘评估的手术需求。快速分析手术设置期间的保证金条件将大大降低手术费用和后续手术率。迄今为止，THz已被证明在区分癌症和健康组织在正式固定，石蜡嵌入（FFPE）组织的有效，但需要额外的调查，以提供可靠的癌症检测在新鲜切除的组织7。
该协议详细介绍了对从生物库获得的新切除人体组织样本进行THz成像和光谱的步骤。建立在新鲜切除的人类乳腺癌组织的THz应用很少用于发表的研究77，18，22，23，18,22,23特别是由研究组不与医院整合。使用新切除组织在其他癌症应用中同样罕见，大多数非乳腺癌的例子都报道了结肠癌24，25。24,原因之一是FFPE组织块比新切除的组织更容易获得和处理，除非用于研究的THz系统是手术工作流程的一部分。同样，大多数商业实验室THz系统不准备处理新鲜组织，而那些确实还处于使用细胞系生长阶段或刚刚开始查看动物模型中切除的组织。要将 THz 应用于术中设置，需要提前为新鲜组织开发成像和表征步骤，以便分析不会干扰执行标准病理学的能力。对于并非本质上意味着术中应用的应用，新鲜组织的表征仍然是一个具有挑战性的步骤，必须加以解决，以在体内应用和分化方面发挥作用。
这项工作的目的是为使用商业 THz 系统的新切除组织 THz 应用提供指南。该协议是在THz成像和光谱系统26为鼠乳腺癌肿瘤13，17，19，17,并13扩展到人类手术组织从生物组197,7，18。虽然该协议是为乳腺癌生成的，但相同的概念可以应用于类似的THz成像系统和其他类型的固体肿瘤癌症，这些癌症在手术中得到治疗，而手术的成功取决于保证金评估27。由于新切除组织上已公布的 THz 结果相当少，这是作者所知的第一部专注于 THz 成像和表征新鲜组织处理协议的工作。
Protocol
该协议遵循阿肯色大学环境健康和安全部门制定的所有要求。 1. 设置组织处理区域 取一个不锈钢金属托盘，用生物危害袋盖住它，如图1所示。任何生物组织处理都将在托盘区域（即组织处理区域）内进行。 在托盘周围准备实验室钳、纸巾、纸巾、滤纸包、组织染料瓶、漂白瓶和乙醇瓶，以便在需要时方便携带。在生物危害物质表面保留…
Representative Results
图9中介绍了根据上述人类乳腺癌肿瘤标本#ND14139从生物库接收的THz成像结果。根据病理报告，#ND14139肿瘤是I/II级渗透导管癌（IDC），通过左乳房结块切除术手术从一名49岁的妇女那里获得。肿瘤的照片如图9A所示，图9B中的病理图像，以及协议中使用方程（1）获得的THz功率光谱图像，如图9C?…
Discussion
新鲜组织的有效THz反射成像主要取决于两个关键方面：1） 正确考虑组织处理（第 2 节和第 4.15 节）;和 2） 舞台设置（主要是第 4.11 节）。由于 DMEM 和其他液体的高反射，组织干燥不足会导致反射增加，无法可视化区域。同时，与成像窗口的不良组织接触会在 THz 反射图像中产生环或低反射点，从而掩盖结果。应作出额外努力，确保组织与成像窗口有良好的接触，包括重新定位组织以获得更好的?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作由国家卫生研究院（NIH）奖（R15CA208798）资助，部分由美国国家科学基金会（NSF）奖1408007资助。脉冲THz系统的资金是通过NSF/MRI奖1228958获得的。我们承认使用国家疾病研究交流会 （NDRI） 在 NIH 赠款 U42OD11158 的支持下采购的纸巾。我们还感谢与俄克拉荷马州立大学俄克拉荷马动物疾病诊断实验室合作，对本工作处理的所有组织进行组织病理学程序。
Materials
| 70% isopropyl alcohol | VWR | 89108-162 | Contains 70% USP grade isopropanol and 30% USP grade deionized water |
| Alconox powder detergent | VWR | 21835-032 | Concentrated detergent to remove organic contaminants from glass, metal, stainless steel, porcelain, ceramic, plastic, rubber, and fiberglass |
| Bio Hazard Bags | Fisher Scientific | 19-033-712 | Justrite FM-Approved Biohazard Waste Container Replacement Bags |
| Cardboard holder | N/A | N/A | Scrap cardboard to keep tissue imaging face intact when immersed in formalin |
| Centrifuge Tubes | VWR | 10026-078 | Centrifuge Tubes with Flat Caps, Conical-Bottom, Polypropylene, Sterile, Standard Line |
| Cotton Swabs | Walmart | 551398298 | Q-tips Original Cotton Swabs used to dye the tissue |
| Ethyl Alcohol | VWR | 71002-426 | KOPTECH Pure (undenatured) anhydrous (200 proof/100%) ethyl alcohol |
| Eye protection goggles | VWR | 89130-918 | Kimberly-clark professional safety glasses |
| Face Mask | VWR | 95041-774 | DUKAL Corporation surgical masks |
| Filter paper | Sigma Aldrich | Z240087 | Whatman grade 1 cellulose filters |
| Formalin solution | Sigma Aldrich | HT501128-4L | 10% neutral buffered formalin |
| Human freshly excised tumors (Infilterating Ductal Carcinoma (IDC)) | National Disease Research Interchange (NDRI biobank | N/A | A protocol is signed with the NDRI for the type of tumors required |
| IRADECON Bleach solution | VWR | 89234-816 | Pre-diluted Sodium Hypochlorite Bleach solution |
| KIMTECH SCIENCE wipes | VWR | 21905-026 | Kimberly-clark professional Kim wipes |
| Laboratory Coat | VWR | 10141-342 | This catalog number is for medium size coat |
| Laboratory tweezers/Forceps | VWR | 82027-388 | Any laboratory tweezers can be used as long as it does not damage the tissue |
| Liquid sample holder (two quartz windows with a 0.1 mm teflon spacer) | TeraView, Ltd | N/A | 1" diameter, and 0.1452" thick quartz windows |
| Nitrile hand gloves | VWR | 82026-426 | This catalog number is for medium size gloves |
| Nitrogen cylinder | Airgas | NI UHP300 | NITROGEN UHP GR 5.0 SIZE 300 |
| Paper towel | VWR | 14222-321 | 11 x 8.78" Sheets, 1 Ply |
| Parafilm | VWR | 52858-076 | Flexible thermoplastic. Rolled, waterproof sheet interwound with paper to prevent self-adhesion. |
| Petri Dish | VWR | 470210-568 | VWR Petri Dish, Slippable, Mono Plate (undivided bottom) |
| Polystyrene Plate | Home Depot | 1S11143A | ~ 10 x 10 cm square piece cut from a 11" x 14" x 0.05" Non-glare styrene sheet |
| ScanAcquire Software | TeraView, Ltd | N/A | System Software for THz reflection imaging measurements |
| Stainless steel low-profile blade (#4689) | VWR | 25608-964 | Tissue-Tek Accu-Edge Disposable Microtome Blades |
| Stainless steel metal tray | Quick Medical | 10F | Polar Ware Stainless Steel Medical Instrument Trays |
| Tissue Marking Dyes | Ted Pella, Inc | Yellow Dye #27213-1 Red Dye #27213-2 Blue Dye #27213-4 |
Used to orient excised tissue samples sent to the histopathology laboratory |
| TPS Spectra 3000 | TeraView, Ltd | N/A | THz imaging and spectroscopy system |
| TPS Spectra Software | TeraView, Ltd | N/A | System Software for THz transmission spectroscopy measurements |
Riferimenti
- Burford, N. M., El-Shenawee, M. O. Review of terahertz photoconductive antenna technology. Optical Engineering. 56 (1), 010901 (2017).
- Sun, Q., et al. Recent advances in terahertz technology for biomedical applications. Quantitative Imaging in Medicine and Surgery. 7 (3), 345-355 (2017).
- Wilmink, G. J., et al. In vitro investigation of the biological effects associated with human dermal fibroblasts exposed to 2.52 THz radiation. Lasers in Surgery and Medicine. 43 (2), 152-163 (2011).
- Arbab, M. H., et al. Terahertz spectroscopy for the assessment of burn injuries in vivo. Journal of Biomedical Optics. 18 (7), 077004 (2013).
- Sy, S., et al. Terahertz spectroscopy of liver cirrhosis: investigating the origin of contrast. Physics in Medicine and Biology. 55 (24), 7587-7596 (2010).
- Yu, C., Fan, S., Sun, Y., Pickwell-Macpherson, E. The potential of terahertz imaging for cancer diagnosis: A review of investigations to date. Quantitative Imaging in Medicine and Surgery. 2 (1), 33-45 (2012).
- El-Shenawee, M., Vohra, N., Bowman, T., Bailey, K. Cancer detection in excised breast tumors using terahertz imaging and spectroscopy. Biomedical Spectroscopy and Imaging. 8 (1-2), 1-9 (2019).
- Yamaguchi, S., et al. Brain tumor imaging of rat fresh tissue using terahertz spectroscopy. Scientific Reports. 6 (30124), 1-6 (2016).
- Rong, L., et al. Terahertz in-line digital holography of human hepatocellular carcinoma tissue. Scientific Reports. 5 (8445), 1-6 (2015).
- Park, J. Y., Choi, H. J., Nam, G., Cho, K., Son, J. In Vivo Dual-Modality Terahertz / Magnetic Resonance Imaging Using Superparamagnetic Iron Oxide Nanoparticles as a Dual Contrast Agent. IEEE Transactions on Terahertz Science and Technology. 2 (1), 93-98 (2012).
- Ji, Y. B., et al. Feasibility of terahertz reflectometry for discrimination of human early gastric cancers. Biomedical Optics Express. 6 (4), 1413-1421 (2015).
- Bowman, T., et al. A Phantom Study of Terahertz Spectroscopy and Imaging of Micro- and Nano-diamonds and Nano-onions as Contrast Agents for Breast Cancer. Biomedical Physics and Engineering Express. 3 (5), 055001 (2017).
- Chavez, T., Bowman, T., Wu, J., Bailey, K., El-Shenawee, M. Assessment of Terahertz Imaging for Excised Breast Cancer Tumors with Image Morphing. Journal of Infrared, Millimeter, and Terahertz Waves. 39 (12), 1283-1302 (2018).
- Bowman, T. C., El-Shenawee, M., Campbell, L. K. Terahertz Imaging of Excised Breast Tumor Tissue on Paraffin Sections. IEEE Transactions on Antennas and Propagation. 63 (5), 2088-2097 (2015).
- Bowman, T., El-Shenawee, M., Campbell, L. K. Terahertz transmission vs reflection imaging and model-based characterization for excised breast carcinomas. Biomedical Optics Express. 7 (9), 3756-3783 (2016).
- Bowman, T., Wu, Y., Gauch, J., Campbell, L. K., El-Shenawee, M. Terahertz Imaging of Three-Dimensional Dehydrated Breast Cancer Tumors. Journal of Infrared, Millimeter, and Terahertz Waves. 38 (6), 766-786 (2017).
- Bowman, T., et al. Pulsed terahertz imaging of breast cancer in freshly excised murine tumors. Journal of Biomedical Optics. 23 (2), 026004 (2018).
- Bowman, T., Vohra, N., Bailey, K., El-Shenawee, M. Terahertz tomographic imaging of freshly excised human breast tissues. Journal of Medical Imaging. 6 (2), 023501 (2019).
- Vohra, N., et al. Pulsed Terahertz Reflection Imaging of Tumors in a Spontaneous Model of Breast Cancer. Biomedical Physics and Engineering Express. 4 (6), 065025 (2018).
- Jacobs, L. Positive margins: the challenge continues for breast surgeons. Annals of Surgical Oncology. 15 (5), 1271-1272 (2008).
- Moran, M. S., et al. Society of Surgical Oncology–American Society for Radiation Oncology Consensus Guideline on Margins for Breast-Conserving Surgery With Whole-Breast Irradiation in Stages I and II Invasive Breast Cancer. International Journal of Radiation Oncology. 88 (3), 553-564 (2014).
- Fitzgerald, A. J., et al. Terahertz Pulsed Imaging of human breast tumors. Radiology. 239 (2), 533-540 (2006).
- Ashworth, P. C., et al. Terahertz pulsed spectroscopy of freshly excised human breast cancer. Optics Express. 17 (15), 12444-12454 (2009).
- Doradla, P., Alavi, K., Joseph, C., Giles, R. Detection of colon cancer by continuous-wave terahertz polarization imaging technique. Journal of Biomedical Optics. 18 (9), 090504 (2013).
- Reid, C. B., et al. Terahertz pulsed imaging of freshly excised human colonic tissues. Physics in Medicine and Biology. 56 (1), 4333-4353 (2011).
- . Teraview.com Available from: https://teraview.com (2019)
- Orosco, R. K., et al. Positive Surgical Margins in the 10 Most Common Solid Cancers. Scientific Reports. 8 (1), 1-9 (2018).
- Bowman, T., et al. Statistical signal processing for quantitative assessment of pulsed terahertz imaging of human breast tumors. 2017 42nd International Conference on Infrared, Millimeter, and Terahertz Waves (IRMMW-THz). , 1-2 (2017).
- Gavdush, A. A., et al. Terahertz spectroscopy of gelatin-embedded human brain gliomas of different grades: a road toward intraoperative THz diagnosis. Journal of Biomedical Optics. 24 (2), 027001 (2019).
Citazione di questo articolo
Vohra, N., Bowman, T., Bailey, K., El-Shenawee, M. Terahertz Imaging and Characterization Protocol for Freshly Excised Breast Cancer Tumors. J. Vis. Exp. (158), e61007, doi:10.3791/61007 (2020).