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非侵入性检测和定量是生物医学研究许多领域中具有挑战性的重要问题。大多数方法依赖于提取少量血液样本，随后使用诸如常规流式细胞术、血细胞计数和微流体分析等技术进行分析。最近，“体内流式细胞术”已经成为一种用于在活体动物中计数荧光标记细胞的技术，而不需要提取血液样品，但是询问体积很小（每分钟约1- 55 L），因此很难检测非常罕见的细胞。因此，需要新的技术用于非常罕见的循环细胞类型的体内研究。 在这个项目中，我们建议开发一个高度新颖的仪器的基础上的荧光层析检测的荧光标记细胞的原则。该仪器采用小型化的高速断层环设计，将适合动物的小肢体（如前肢，后腿或尾巴），并允许血液流速高达0.2至0.5 mL/min的极灵敏检测，因此小鼠的全部血液体积将在不到10分钟内采样。为了实现这一点，该系统将用途：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）组织和细胞自发荧光的有效光谱抑制，以及iv）在100 Hz下的快速、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，首先在具有模拟自体荧光的肢体模拟流动体模模型中，其次在BALB/c小鼠体内使用两组荧光标记细胞系。我们预计，该系统将在生物医学研究的许多领域，包括在体内检测转移性肿瘤细胞和循环造血干细胞的应用。 公共卫生相关性：该项目的目标是开发一种高度新颖的仪器，用于活体动物中非常罕见的循环细胞的非侵入性检测和定量。该仪器将使用高速小型化断层环来检测循环中的单个荧光标记细胞。该环将安装在四肢（如前肢，后腿或尾巴）周围，并允许在不到10分钟的时间内对小鼠的整个血液体积进行采样，提供前所未有的检测灵敏度。我们预计，该系统将在生物医学研究的许多领域，包括在体内检测转移性肿瘤细胞和循环造血干细胞的应用。

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.