在生物环境的新技术的产生和观测化学发光
Summary
This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.
Abstract
Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.
Introduction
在近几十年中，成像技术已经彻底改变，医生诊断和监测疾病的方法。这些成像技术，然而，已经在很大程度上仅限于全身成像系统，例如正电子发射断层扫描（PET），单光子发射计算机断层摄影（SPECT），计算机断层扫描（CT）和磁共振成像（MRI）。特别注意了癌症，而技术突破成像有很大的提高，这种病诊断和治疗的方式。尽管有这些进展，但是一个地方，这些成像技术只是不适合：手术室。虽然全身成像技术可在手术计划帮助下，他们通常缺乏空间分辨率足够高，以帮助医生实时确定是否所有的肿瘤组织已被删除或残留的肿瘤组织仍然在手术切缘1隐藏。确保无浸润肿瘤边缘被留下是最重要的手术目标之一，和外科医生必须走严格而谨慎的组织切除之间的紧绳子。如果太多被去除，不需要的副作用对患者正在加剧;如果太少被删除，复发率增加2，3。因此，划定精确的肿瘤边缘是至关重要的，而且我们相信，化学发光成像术可通过帮助医生形象化恶性组织，否则会没有被发现已建立的技术，以提高肿瘤边缘的识别精度。
目前正在调查其可能的实用程序，术中成像系统的许多影像技术。这些包括β-和γ射线发射探头4，光学荧光5，拉曼光谱6 </sup>，7和切伦科夫发光8,9。迄今为止，然而，没有这些都成为确立为标准的临床工具。光学荧光成像迄今被证明是最有前途的这些技术，因此是最探讨。虽然它已经被证明是对许多应用的有价值的工具，它也不是没有局限性。事实上，它的主要缺点是由固有的自发荧光的生物组织所产生的背景荧光。这样的背景自发荧光信号是周围组织的激励的产物，除了荧光团，通过所需要的荧光信号的产生的外部光源。从实践的角度来看，这自发荧光可以潜在地导致低信号对噪声比，它可以限制在手术室这种技术的效用。
校长优势化学发光成像在荧光成像是没有激发光是必要的。其结果是，没有背景自发荧光。在化学发光成像，代替化学生成的激发能量。这一过程不会产生意外的背景信号，因此可以导致更高的信号 – 噪声比。这可能最终导致手术切缘的更精确和准确的检测。出人意料的是，这种方法作为一种手术成像技术的效用仍然未开发的10。事实上，最近的例子，这种技术是鲁米诺由髓过氧化物酶的小鼠11，12，13的氧化。因此化学发光生物医学成像是研究的一个，而未开发地区，可以具有以下优点：（1）导致与喜低背景信号最小自发荧光gher信号 – 噪声比; （2）化学发光发射范围从可见光到近红外的波长可调波长; （3）官能化的化学发光复合物，当与连接器的技术相结合，有针对性已经存在的生物分子，靶向分子成像探针14的整个库提供接入。
验证的原理该研究说明了化学发光成像在使用基于钌 – 成像剂的生物医学设置的潜在效用。该化合物的化学发光特性很好的研究，以调查可追溯至60年代中期15。经化学活化，所述试剂产生在约600毫微米16的光，这是非常适合用于医疗成像目的。活化能是由导致激发态-其具有650纳秒寿命在水中17 -foll的氧化还原反应提供在此激发态的松弛由光子的产生欠款。通过使用一个特别设计的远程喷雾器的，我们能够检测到化合物既体外 和体内 。最初的实验的结果是非常有前景的，这表明该技术的进一步调查。
Protocol
Representative Results
Discussion
这里，我们已经提出了一种技术，能够通过由化学发光记者创建的光子的发射光学划定组织。相对于其他，更成熟的，技术4，5，6，7，8，9，此化学发光报道系统采用了成像探针是无放射性和在非常高的灵敏度水平便于检测。或许更重要的是，化学…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank Prof. Jan Grimm and Mr. Travis Shaffer for their helpful discussions and Mr. David Gregory for editing the manuscript. Technical services provided by the MSK Animal Imaging Core Facility, supported in part by NIH Cancer Center Support Grant P30CA008748-48, are gratefully acknowledged. The authors thank the NIH (K25 EB016673 and R21 CA191679, T.R. and 4R00CA178205-02, B.M.Z.), the MSK Center for Molecular Imaging and Nanotechnology (T.R.), the Tow Foundation (B.C.), and the National Science Foundation Integrative Graduate Education and Research Traineeship (IGERT 0965983 at Hunter College for B.C. and T.M.S.) for their generous support. The research reported in this publication was supported by funding from the King Abdullah University of Science and Technology.
Materials
| Wood part A (12.5×2.5×1.8 cm) | Woodcraft | 131404 | Cut from a 3/4” x 24” x 30” birch plywood sheet |
| Wood part B (12.7×10.7×1.8cm) | Woodcraft | 131404 | Cut from a 3/4” x 24” x 30” birch plywood sheet |
| Wood part C (11×2.5×1.8cm) | Woodcraft | 131404 | Cut from a 3/4” x 24” x 30” birch plywood sheet |
| Screws (4×25 mm) | Screwfix | 79939 | |
| Harmon Face Values 3oz mini sprayer | Bed, Bath and Beyond | ||
| stainless steel rod (10 cm of 1/16” steel) | Metals Depot Int. Inc. | 2192 | |
| Pencil Classic HB | Papermate | 58592 | |
| Paper clip | Office Depot | 221720 | |
| speaker cable | RCA Inc. | AH1650SN | |
| Energizer 9V alkaline battery | Energizer Holdings Inc. | EN22 | |
| Hitech HS-82MG Micro Servo Motor, 3.4kg/cm output torque @ 6V | Hitech RCD USA Inc. | 32082S | |
| Name | Company | Catalog Number | Comments |
| 28 cm plastic cable ties | General Electric Inc. | 50725 | |
| Duct tape | 3M Inc. | 3939 | |
| littleBits w1 wire | littleBits Inc. | w1 wire | |
| littleBits p1 power | littleBits Inc. | p1 power | |
| littleBits i2 toggle switch | littleBits Inc. | i2 toggle switch | |
| littleBits 011 servo | littleBits Inc. | 011 servo | |
| 20 cm plastic covered wire twist ties | Four Star Plastics | 71TIE8000 | |
| Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate | Sigma-Aldrich Inc. | 224758 | |
| Ammonium cerium(IV) nitrate | Sigma-Aldrich Inc. | 22249 | |
| Isofluorane | Baxter Healthcare | 1001936060 | |
| PBS | Sigma-Aldrich | PBS1 | |
| Ethanol | Sigma-Aldrich | 2854 | |
| Triethylamine | Sigma-Aldrich Inc. | T0886 | |
| Water | Water was purified using a Milipore Mili-Q (R ≥ 18 MΩ) | ||
| Female nude (outbred) mice | Jackson Laboratories | 1929 | age 5 – 6 weeks |
| Strain C57BL/6J | |||
| NU/J male mice at | Jackson Laboratories | 2019 | age 6 – 8 weeks |
| IVIS 200 bioluminescence reader | Caliper Live Science | ||
| Live Image 4.2 software | Perkin-Elmer | 128165 | |
| Microscope slides | ThermoScientific | 4951PLUS4 |
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