TomoFlow: Tomographic Imaging Two-Photon Flow Cytometry

多光子显微镜允许深度光学穿透，使其成为组织光学成像的首选方法。为了了解疾病的分子和细胞起源，在高时空分辨率和相关尺度上感知和可视化亚细胞单位和细胞间相互作用至关重要。此外，为了获得稳健可靠的统计数据，有必要研究大量异质细胞群。这可以通过成像流式细胞术（IFC）最佳地解决，利用微流体中细胞的显微成像。在这里，我们提出了一种高通量的IFC技术，利用高速双光子显微镜和体积IFC进行三维采集，用于研究细胞群和类器官。双光子显微镜的高穿透性和体积采集导致层析成像采集。因此，我们发明了这个新的技术平台TomoFlow，用于层析成像。高通量是由最近推出的衍射激发（SLIDE）技术的光谱时间激光成像，提供每秒4000帧的成像速率。对于TomoFlow，我们将开发新的微流控流芯片，以实现高三维分辨率（< 1µm3）的层析成像。我们将进一步将TomoFlow技术应用于全血悬浮液中肿瘤细胞的检测、类器官和组织碎片成像以及类器官的药理学研究，在细胞和亚细胞水平上具有高分辨率和高通量，具有丰富的统计数据。这项研究有可能为研究疾病的细胞起源提供一种新的生物医学诊断工具，并在细胞和分子水平上开发靶向、个性化的治疗和药物。

Multiphoton microscopy allows deep optical penetration making it the method of choice for optical imaging in tissue. In order to understand the molecular and cellular origin of disease, it is crucial to sense and visualize sub-cellular units and cell-to-cell interactions at high temporal and spatial resolution and at relevant scales. In addition, for a robust and reliable statistic it is necessary to study a large, heterogenous population of cells. This can be optimally addressed by imaging flow cytometry (IFC), employing microscopic imaging of cells in microfluidic flow. Here, we propose a high throughput IFC technique with three-dimensional (3D) acquisition for the study of cell populations and organoids by employing high speed Two-Photon microscopy and volumetric IFC. The high penetration of Two-Photon microscopy and the volumetric acquisition lead to a tomographic imaging acquisition. Thus, we coin this new technology platform TomoFlow for tomographic flow imaging. The high-throughput is enabled by the recently introduced spectro-temporal laser imaging by diffractive excitation (SLIDE) technique providing imaging rates of 4.000 frames per second. For TomoFlow, we will develop new microfluidic flow chips to enable tomographic imaging at high three-dimensional resolution (< 1µm3). We will further apply the TomoFlow technology to the detection of tumor cells in flow, organoid and tissue fragment imaging suspended in whole, unfiltered blood as well as pharmacological studies on organoids with high-resolution at the cellular and sub-cellular level and high throughput for rich statistics. This research endeavor has the potential of providing a new biomedical diagnostic tool for the study of the cellular origin of disease and the development of targeted, personalized therapies and medicine on the cellular and molecular level.