Development of Instrumentation for Fluorescence-Guided Surgery

用于体内使用的分子靶向探针的快速发展导致了对结合来自这些探针的信息的临床应用的日益增长的兴趣。特别地，需要在手术期间使这些靶向探针可视化的技术，特别是识别用于移除或保存的组织。 当观察组织中的荧光探针时，主要困难通常是将探针荧光与组织自体荧光分离。由于自发荧光具有与荧光团不同的光谱特性，因此可以使用多光谱成像容易地将其分离，其中可以针对每个像素拍摄几个完整的发射光谱。虽然声光可调谐滤波器的实现大大提高了这些技术的速度，但获取图像立方体所需的最小时间对于商业上可用的系统仍然是几秒钟。 当使用效率较低的荧光探针成像时或当以较低剂量使用时，图像采集时间可以长达几分钟。这种数据采集速率使得在外科手术过程中使用多光谱成像来提供实时反馈是不切实际的。 即使当用于诊断目的时，在获取图像立方体所需的时间期间对象的不可避免的运动也可能导致空间分辨率的不可接受的损失。 该项目的重点是仪器的发展，将荧光和多光谱成像到外科手术中的应用，使用两种主要的方法。 第一种方法是开发一种荧光探针实时可视化系统，以指导手术。 第二种方法是开发用于诊断应用的系统，以提供用于多光谱图像立方体的空间配准的白光图像堆栈。 作为多光谱成像的替代方案，对发射光和激发光的积极过滤可以帮助最小化组织自体荧光的影响。虽然随着光谱窗口变窄，信号电平会下降，但这可以通过使用灵敏的相机来克服，例如冷却CCD，ICCD，甚至EMCCD。由于使用单个光谱窗口，因此可以大大加快数据采集速度。该仪器的直接应用是使用NCI开发的GSA-罗丹明绿色探针识别卵巢癌腹膜转移。 第一个原型仪器，用于模拟细胞减灭术在小鼠模型的卵巢癌，有足够的灵敏度，以识别标记的转移，在图像采集速率为每秒5帧的特异性与多光谱系统实现的。 一种改进的原型，允许以每秒15帧的速度采集图像，灵敏度提高，用于麻醉动物的细胞减灭术，取得了与先前模拟手术相似的成功。 今年，我们还继续开发一个单独的多光谱系统，使用一个92：8分束器和一个与多光谱仪器并行的低成本单色CCD相机，为多光谱图像立方体的物理配准提供一个白色光图像堆栈。 子宫颈癌动物模型的初步实验正在进行中。 为该应用开发的硬件可以容易地适于将第二相机结合到用于荧光引导手术的仪器中;该第二相机可以用于第二荧光图像，以便提供简单的自体荧光校正，或者用于手术野的伪白光图像。 最后，使用上转换器进行了一些初步的原理验证体内成像实验，在这种情况下，上转换纳米晶体吸收980 nm的光并在可见光至近红外线中发射。 这些新化合物可以允许单波长成像，没有可检测的自发荧光;组织背景是如此之低，以至于可能需要白色光图像来提供解剖参考。虽然在开发这些纳米粒子用于医疗应用方面仍有相当多的工作要做，但这是未来努力的一个有希望的途径。

The rapid development of molecularly targeted probes for use in vivo has led to growing interest in clinical applications that incorporate information from these probes. In particular, there is a need for techniques to visualize these targeted probes during surgery, particularly to identify tissue either for removal or preservation. When looking at fluorescent probes in tissue, the major difficulty is usually separating the probe fluorescence from the tissue autofluorescence. Because the autofluorescence has different spectral properties than the fluorophore, it can easily be separated using multispectral imaging, in which several full emission spectra can be taken for each pixel. Although the implementation of acousto-optic tunable filters has greatly increased the speed of these techniques, the minimum time required to acquire an image cube is still several seconds with commercially available systems. When imaging using fluorescent probes with lower efficiency or when used at lower doses, the image acquisition time can be as long as several minutes. This data acquisition rate makes the use of multispectral imaging to provide real-time feedback during a surgical procedure impractical. Even when used for diagnostic purposes, the unavoidable motion of the subject during the time required to acquire an image cube can lead to an unacceptable loss of spatial resolution. This project focuses on the development of instrumentation for incorporating fluorescent and multi-spectral imaging into surgical applications, using two major approaches. The first approach is to develop a system for real-time visualization of fluorescent probes to guide surgery. The second approach is to develop a system for diagnostic applications, to provide a white-light stack of images for spatial registration of the multispectral image cube. As an alternative to multispectral imaging, aggressive filtering of both emission and excitation light can help to minimize the effects of tissue autofluorescence. Although the signal level drops as the spectral window is narrowed, this can be overcome by using a sensitive camera, such as a cooled CCD, ICCD, or even an EMCCD. Because a single spectral window is used, substantially faster data acquisition is possible. The immediate application for this instrument was the identification of peritoneal metastases in ovarian cancer, using a GSA-Rhodamine Green probe developed in NCI. The first prototype instrument, used for mock cytoreductive surgery in a murine model of ovarian cancer, had sufficient sensitivity to identify labeled metastases at an image acquisition rate of five frames per second with specificity comparable to that achieved with a multispectral system. An improved prototype, which permitted image acquisition at fifteen frames per second with enhanced sensitivity, was used for cytoreductive surgery on anesthetized animals with similar success to the previous mock surgery. This year, we also continued development on a separate multispectral system to provide a white light image stack for physical registration of the multispectral image cube, using a 92:8 beam splitter and a low-cost monochrome CCD camera in parallel with the multispectral instrumenation. Preliminary experiments on a cervical cancer animal model are underway. The hardware developed for this application could be easily adapted to incorporate a second camera into the instrument for fluorescence guided surgery; this second camera could be used either for a second fluorescence image, in order to provide a simple autofluorescence correction, or for a pseudo-white light image of the surgical field. Finally, some initial proof-of-principle in vivo imaging experiments were performed using upconverters, in this case upconverting nanocrystals that absorb 980 nm light and emit in the visible to near-infrared. These novel compounds could allow single-wavelength imaging with no detectable autofluorescence; the tissue background is so low that a white light image will likely be required to provide anatomical reference. Although considerable work remains in developing these nanoparticles for medical applications, this is a promising avenue for future efforts.