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

挑战。肿瘤球体(和有机体)已成为癌症研究的工具。这些 自组织三维(3D)系统可以概括患者的表型和功能特征 体内肿瘤，从而成为研究肿瘤异质性的强大试验床，与 环境(如细胞外基质)和对外界刺激(如化疗、放射)的反应。完全 然而，由于缺乏高通量的分析方法，利用球体的效用受到阻碍。传统型 虽然Bright-field显微镜被广泛用于监测培养物中的球体，但它不能捕捉到详细的细胞 组织；先进的fl荧光显微镜可以分辨单个细胞，但其成像吞吐量 受小fi视野(FOV)和扫描机制的限制。创新。我们的目标是 提出了一种用于肿瘤球体单细胞分析的新型体积成像显微镜(VIM)。SPECIfiCALY， 我们将探索将傅里叶层析显微镜(FPM)与衍射层析成像相结合。FPM是基于 在空间编码照明技术上，采集低分辨率图像序列，同时改变 点光源的位置。然后，这些图像在傅里叶空间中进行数值组合，这 允许fi在2D图像中实现宽视野和高空间分辨率。我们认为全3D 在数值重建过程中，通过考虑光学衍射，可以恢复显微图像。 接近了。目标1.系统开发。我们将建立一个具有以下特点的VIM系统：i)一种新的数值算法 重建三维体图像；ii)新的光照策略，以加快数据采集；iii) 针对球体培养和药物治疗进行了优化的MicroflUIDIC墨盒；以及iv)多色成像能力 分子检测。完整的vim将以高分辨率解析构成球体的单个细胞。 (横向，0.4微米；轴向，1微米)，成像体积大。目的2.肿瘤球体治疗监测。我们 将测试Vim的实用价值：启用Vim的椭球体成像将比批量成像更早地揭示 球体对药物治疗有反应或耐药。为了生成肿瘤模型，我们将使用原代GBM 来自病人的细胞。基底膜球体将被培养，并在微fl治疗中使用药物(替莫唑胺) 子弹。我们将使用VIM来监测单个细胞如何在治疗下改变其表型，以及 将这些变化与治疗结果相关联。冲击力。VIM将成为治疗癌症的变革性工具 研究，为研究人员提供丰富的数据集和非常先进的分析。立马 应用包括更好地监测3D细胞培养中的抗癌药物反应，分析细胞 异质性，并前瞻性地检测不同生理条件下的细胞命运。这些 结果将加强肿瘤球体在癌症研究中的临床和科学应用。

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.