Three-dimensional fluorescence imaging flow cytometry at up to million frames per second

每秒高达百万帧的三维荧光成像流式细胞术

• 批准号: 10568627
• 负责人: ADELA BEN-YAKAR
• 金额: $ 41.55万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-15 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10568627
• 关键词: 3-Dimensional; Animals; Big Data; Biological; Biological Assay; Caenorhabditis elegans; Cancer Biology; Cardiac; Cardiotoxicity; Cells; Collaborations; Color; Communities; Cytometry; Data; Data Analyses; Data Compression; Data Display; Data Reporting; Detection; Development; Diagnosis; Early Diagnosis; Emulsions; Flow Cytometry; Frequencies; Goals; Hela Cells; Hematology; Image; Immersion; Immunology; Individual; Infrastructure; Inherited; Label; Length; Light; Lobe; Location; Measurement; Measures; Metadata; Methods; Microbiology; Microfluidic Microchips; Microscope; Microscopy; Morphologic artifacts; Morphology; Names; Nature; Noise; Optics; Organoids; Outcomes Research; Pathology; Patients; Pharmaceutical Preparations; Phenotype; Population; Resolution; Rotation; Sample Size; Sampling; Scanning; Scientist; Side; Signal Transduction; Silicon; Spatial Distribution; Specimen; Speed; Spottings; System; Technology; Therapeutic; Three-Dimensional Imaging; Time; Tissue Engineering; Toxic effect; Training; Translations; Visualization; absorption; cancer cell; cancer diagnosis; cell dimension; cellular imaging; computer infrastructure; convolutional neural network; data acquisition; data handling; deep learning model; detection method; drug discovery; experimental study; fluorescence imaging; heart dimension/size; high resolution imaging; high reward; high risk; imaging platform; improved; meter; microscopic imaging; next generation; novel; photomultiplier; rare cancer; screening; stem cell biology; submicron; tool; two photon microscopy; two-dimensional; two-photon

Flow cytometry is the tool of choice for high-speed analysis of large cell populations, with the tradeoff of lacking intracellular spatial information. Imaging flow cytometry (IFC) has emerged as a new tool that combines advantages of microscopy with the high speed of flow cytometry. However, they can only provide 2D images to determine three-dimensional (3D) distribution of cellular features, have a limited field of view (FOV), and require precise control of the fluidic system to minimize image blurring due to uncontrolled cell rotation or translation across the FOV. The absence of 3D imaging results in ambiguity of object locations and blurring by focal depth due to the projection of a 3D cell into a 2D image. Although in the last decades flow cytometry systems that can actually acquire three-dimensional (3D) spatial information were developed, constraints related to resolution and samples size remained as their biggest limitation. Therefore, the goal of this proposal is to develop the next generation 3D imaging flow cytometers with high-throughput and high-content capabilities for 3D imaging of hundreds to thousands of cells and spheroids per second with high resolution, for the first time. We propose to develop such a cytometry method, using a novel microscopy method, Line Excitation Array Detection microscopy (LEAD), that can image objects in large field of views at the rate of current 1D cytometers, but with high 3D resolution and high signal-to-noise ratios (SNR). Our proposed LEAD cytometer is a fast-scanned light-sheet microscope capable of MHz frame rates. We will develop the fastest MHz line-scanning method using a longitudinal acousto-optic deflector driven by a chirped frequency signal. We will image the scanned light sheet using a linear silicon photomultiplier array, which will provide the sensitivity required when scanning so quickly, and the parallel readout required for such high frame rates. First, we will develop linear LEAD 3D imaging flow cytometry at sub-micron scale resolution and small FOVs. Although our preliminary data indicates we will be able to image at 100 kHz – MHz frame rates at such high resolution with high SNR, we will perform experiments measuring the SNR to determine the operating range of LEAD cytometry. In the second aim, we will increase the FOV by developing two-photon LEAD imaging flow cytometry with Bessel beams. To support the larger FOV, we will develop a 128-channel data acquisition system using eight 16-channel data acquisition cards. In the third aim, we will develop a state-of-the-art computational infrastructure that allows for file transfers up to 25 GB/s, storage (>100 TB), and analysis that only takes 3x the imaging time. We will use 2 deep learning models for analysis. If successful, this high-risk/high-reward proposal would alter the imaging flow cytometry landscape. The proposed 3D imaging flow cytometer can offer improved cell and spheroid analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. Improved sensitivity will help users to improve research outcomes or diagnose patients with higher statistical power.

流式细胞术是高速分析大细胞群的首选工具，但缺点是缺乏 细胞内空间信息成像（IFC）已成为一种结合了细胞内空间信息的新工具。 显微镜具有流式细胞术高速的优点，但它们只能提供 2D 图像。 确定细胞特征的三维 (3D) 分布，具有有限的视野 (FOV)，并且需要 精确控制流体系统，最大限度地减少由于不受控制的细胞旋转或平移而导致的图像模糊 缺乏 3D 成像会导致物体位置模糊以及焦深模糊。 由于将 3D 细胞投影到 2D 图像中，尽管在过去几十年中流式细胞术系统可以做到这一点。 实际获取三维（3D）空间信息的开发，与分辨率和相关的约束 样本大小仍然是其最大的限制，因此，该提案的目标是开发下一个。 新一代 3D 成像流式细胞仪，具有高通量和高内涵功能，可用于 3D 成像 我们首次提出每秒数百至数千个高分辨率的细胞和球体。 开发这样的细胞计数方法，使用一种新颖的显微镜方法，线激发阵列检测显微镜 (LEAD)，可以以当前 1D 细胞仪的速率对大视野中的物体进行成像，但具有高 3D 我们提出的 LEAD 细胞仪是一种快速扫描的光片。 我们将使用具有 MHz 帧速率的显微镜开发最快的 MHz 线扫描方法。 由啁啾频率信号驱动的纵向声光偏转器我们将对扫描的光片进行成像。 使用线性硅光电倍增管阵列，这将提供快速扫描时所需的灵敏度， 首先，我们将开发线性 LEAD 3D 成像流程。 尽管我们的初步数据表明我们将实现亚微米级分辨率和小视场的细胞计数。 能够以 100 kHz – MHz 帧速率、如此高分辨率和高信噪比进行成像，我们将进行实验 测量 SNR 以确定 LEAD 细胞计数的操作范围 在第二个目标中，我们将增加。 使用贝塞尔光束的双光子 LEAD 成像流式细胞仪的 FOV 支持更大的开发 FOV， 我们将使用八块16通道数据采集卡开发一个128通道数据采集系统。 我们的目标是开发最先进的计算基础设施，文件传输速度高达 25 GB/s， 存储（> 100 TB），分析只需三倍的成像时间，我们将使用 2 个深度学习模型。 如果成功，这一高风险/高回报的提议将改变成像流式细胞术的格局。 所提出的 3D 成像流式细胞仪可以在多种生物医学领域提供改进的细胞和球体分析 癌症生物学、微生物学、免疫学、血液学和干细胞生物学等领域提高灵敏度。 将帮助用户改善研究结果或以更高的统计能力诊断患者。