Tomographic In Vivo Flow Cytometer for Counting Rare Circulating Cells

描述(申请人提供)：活体动物稀有循环细胞的非侵入性检测和量化在许多生物医学研究领域是一个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，然后在稍后使用常规的流式细胞仪、血细胞分析仪和微流控分析等技术进行分析。最近，“活体流式细胞术”作为一种不需要提取血液样本的活体动物荧光标记细胞计数技术出现了，但询问的体积很小(每分钟约1-55L)，因此很难检测到非常稀有的细胞。因此，需要新的技术对非常罕见的循环细胞类型进行体内研究。在这个项目中，我们计划开发一种基于荧光断层扫描检测荧光标记细胞的原理的高度新颖的仪器。该仪器采用了微型高速断层扫描环设计，可以安装在动物的小腿(如前肢、后腿或尾巴)周围，并允许以高达每分钟0.2至0.5毫升的血液流速进行极其灵敏的检测，从而在不到10分钟的时间内采集到老鼠的全部血量。为了实现这一目标，该系统将使用：i)调制高强度LED进行荧光激发，ii)高灵敏度光子计数检测发射的荧光光，iii)有效地抑制组织和细胞的自发荧光，以及iv)100赫兹的快速实时图像重建。我们将展示该仪器能够分辨单个循环荧光标记的细胞，首先是在模拟自体荧光的模拟肢体流动模型中，其次是在BALB/c小鼠体内使用两组荧光标记的细胞系。我们预计该系统将在生物医学研究的许多领域得到应用，包括体内检测转移的肿瘤细胞和循环中的造血干细胞。 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量化。该仪器将使用高速微型断层摄影环来检测循环中单个荧光标记的细胞。这种环将被安装在四肢(如前肢、后腿或尾巴)周围，并将在不到10分钟的时间内对老鼠的整个血液容量进行采样，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得到应用，包括体内检测转移的肿瘤细胞和循环中的造血干细胞。

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.