Tomographic In Vivo Flow Cytometer for Counting Rare Circulating Cells

描述（由申请人提供）：活体动物中稀有循环细胞的无创检测和定量在生物医学研究的许多领域是一个具有挑战性和重要的问题。大多数方法依赖于提取少量血液样本，然后使用常规流式细胞术、血细胞计和微流体分析等技术对其进行分析。最近，“体内流式细胞术”作为一种不需要提取血液样本就能在活体动物中计数荧光标记细胞的技术而出现，但检测量很小（每分钟约1-55L），因此很难检测到非常罕见的细胞。因此，需要新的技术来进行非常罕见的循环细胞类型的体内研究。在这个项目中，我们建议开发一种基于荧光标记细胞的荧光层析检测原理的高度新颖的仪器。该仪器采用小型化的高速断层扫描环设计，可安装在动物的小肢体（如前肢，后肢或尾巴）周围，并允许极其敏感的检测，血液流速高达每分钟0.2至0.5 mL，因此在不到10分钟的时间内即可采样小鼠的整个血量。为了实现这一目标，该系统将使用：i)用于荧光激发的调制高强度led， ii)发射荧光的高灵敏度光子计数检测，iii)组织和细胞自身荧光的有效光谱抑制，以及iv) 100Hz的快速实时图像重建。我们将证明该仪器能够分辨单个循环荧光标记细胞，首先在模拟自身荧光的肢体模拟流动幻影模型中，其次在体内使用两组荧光标记细胞系的BALB/c小鼠中。我们预计该系统将在生物医学研究的许多领域得到应用，包括体内检测转移性肿瘤细胞和循环造血干细胞。

DESCRIPTION (provided by applicant): Non-invasive detection and quantification of rare circulating cells in live animals is a challenging and important problem in many areas of biomedical research. Most methods rely on the extraction of small blood samples which are analyzed at a later time using techniques such as conventional flow cytometry, hemocytometry and micro-fluidic analysis. Recently, 'in vivo flow cytomety' has emerged as a technique for counting fluorescently-labeled cells in live animals without the need for extracting blood samples, but the volume of interrogation is small (~1-55L per minute) so that detection of very rare cells is difficult. Therefore, new techniques are needed for the in vivo study of very-rare circulating cell types. In this project, we propose to develop a highly-novel instrument based on the principle of fluorescence tomographic detection of fluorescently-labeled cells. The instrument utilizes a miniaturized high-speed tomographic ring design that will fit around a small limb (such as the forelimb, hindleg or tail) of an animal and allow extremely sensitive detection with blood flow rates up to 0.2 to 0.5 mL per minute, so that the entire blood volume of a mouse will be sampled in less than 10 minutes. To achieve this, the system will use: i) modulated high- intensity LEDs for fluorescence excitation, ii) high-sensitivity photon counting detection of emitted fluorescent light, iii) efficient spectral rejection of tissue and cellular autofluorescence, and iv) rapid, real-time image reconstruction at 100Hz. We will demonstrate that the instrument is capable of resolving single circulating fluorescently-labeled cells, first in a limb- mimicking flow phantom model with simulated autofluorescence, and second in BALB/c mice in vivo using two sets of fluorescently-labeled cell lines. We anticipate that the system will have applications in many areas of biomedical research including in vivo detection of metastatic tumor cells and circulating hematopoietic stem cells. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a highly novel instrument for the non-invasive detection and quantification of very rare circulating cells in live animals in vivo. The instrument will use a high-speed miniaturized tomographic ring to detect single fluorescently-labeled cells in circulation. The ring will be fitted around a limb (such as the forelimb, hindleg or tail) and will allow sampling of the entire blood volume of a mouse in less than 10 minutes, offering unprecedented detection sensitivity. We anticipate that the system will have applications in many areas of biomedical research including in vivo detection of metastatic tumor cells and circulating hematopoietic stem cells.