3D Fourier Imaging System for High Throughput Analyses of Cancer Organoids

挑战肿瘤球状体（和类器官）已成为癌症研究中的工具。这些 自组织的三维（3D）系统可以概括患者的表型和功能特征 肿瘤在体内，从而作为一个强大的测试床，研究肿瘤异质性，相互作用， 环境（例如，细胞外基质），以及对外部刺激的反应（例如，化疗、放疗）。充分 然而，由于缺乏高通量分析方法，利用球体的效用受到阻碍。常规 明场显微镜虽然广泛用于监测培养中的球状体， 先进的荧光显微镜可以分辨单个细胞，但其成像吞吐量 受限于小视场（FOV）和所涉及的扫描机制。创新。我们的目标是 提出了一种新的体积成像显微镜（Vim），用于肿瘤球体中的单细胞分析。具体来说， 我们将探讨整合傅立叶重叠关联显微镜（FPM）与衍射层析成像。FPM基于 在空间编码照明技术上，收集低分辨率图像序列，同时改变 点光源的位置。然后，这些图像在傅立叶空间中进行数字组合， 允许FPM在2D图像中实现宽视场和高空间分辨率。我们认为全3D 通过在数值重建期间考虑光学衍射，可以恢复显微图像。 接近。目标1。系统开发。我们将建立一个Vim系统，其特点是：i）一个新的数值算法 重建3D体积图像; ii）一种新的光照明策略，以加快数据采集; iii） 优化用于球状体培养和药物治疗的微荧光素盒;和iv）用于以下的荧光素成像能力： 分子检测完整的Vim将以高分辨率分辨构成球体的单个细胞 （横向，0.4 µm;轴向，1 µm）。目标二。肿瘤球体的治疗监测。我们 将测试Vim的实际效用：启用VIM的球体成像将比批量成像更早地揭示 球状体对药物治疗有反应或有抗性。为了生成肿瘤模型，我们将使用原发性GBM 患者的细胞。GBM球状体将在微囊内生长并用药物（替莫唑胺）处理 子弹。我们将使用Vim来监测单细胞在治疗下如何改变其表型， 将这些变化与治疗结果联系起来。冲击Vim将成为癌症的变革工具 研究，为研究人员提供丰富的数据集和先进的分析。立即 应用包括在3D细胞培养中更好地监测抗癌药物反应，分析细胞 异质性，并在各种生理条件下前瞻性地检测细胞命运。这些 研究结果将加强肿瘤球体在癌症研究中的临床和科学效用。

Challenges. Tumor spheroids (and organoids) have become an instrumental tool in cancer research. These self-organized, three-dimensional (3D) systems can recapitulate phenotypic and functional traits of patient tumors in vivo, thereby serving as a powerful testing bed to study tumor heterogeneity, interactions with the environment (e.g., extracellular matrix), and responses to external stimuli (e.g., chemotherapy, radiation). Fully harnessing spheroids' utility, however, is stymied by lack of high-throughput analysis methods. Conventional bright-field microscopy, although widely used to monitor spheroids in culture, fails to capture detailed cellular organizations; advanced fluorescent microscopy can resolve individual cells, but its imaging throughput is restricted by the small field-of-view (FOV) and the scanning mechanisms involved. Innovations. We aim to advance a new volumetric imaging microscope (VIM) for single cell analyses in tumor spheroids. Specifically, we will explore integrating Fourier ptychographic microscopy (FPM) with diffraction tomography. FPM is based on a spatially coded-illumination technique, collecting low resolution image sequences while changing the position of a point-light source. These images are then numerically combined in the Fourier space, which allows FPM to achieve both wide field-of-view and high spatial resolution in 2D images. We reason that full 3D microscopic images can be recovered by accounting for optical diffraction during the numerical reconstruction. Approaches. Aim 1. System development. We will build a VIM system featuring: i) a new numerical algorithm to reconstruct 3D volumetric images; ii) a new light-illumination strategy to speed up the data acquisition; iii) microfluidic cartridges optimized for spheroid culture and drug treatment; and iv) multicolor imaging capacity for molecular detection. The complete VIM will resolve individual cells constituting a spheroid at high resolution (lateral, 0.4 µm; axial, 1 µm) in a large imaging volume. Aim 2. Treatment monitoring with tumor spheroids. We will test VIM's practical utility: VIM-enabled spheroid imaging will reveal earlier than bulk imaging whether a spheroid is responsive or resistance to drug treatment. To generate a tumor model, we will use primary GBM cells from patients. GBM spheroids will be grown and treated with drug (temozolomide) inside microfluidic cartridges. We will use the VIM to monitor how single cells change their phenotypes under treatment, and correlate these changes with treatment outcomes. Impact. The VIM will be a transformative tool for cancer research, empowering researchers with rich data sets and substantially advanced analytics. Immediate applications include better monitoring of anticancer drug responses in 3D cell culture, analyzing cellular heterogeneity, and prospectively detecting cellular fate under various physiological conditions. These outcomes will strengthen the clinical and scientific utility of tumor spheroids in cancer research.