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.

流式细胞仪是对大型细胞种群进行高速分析的首选工具，而缺乏的权衡 细胞内空间信息。成像流式细胞术（IFC）已成为一种结合的新工具 显微镜的优势，流式细胞仪的高速。但是，他们只能提供2D图像 确定细胞特征的三维（3D）分布，视野有限（FOV），并且需要 精确控制流体系统，以最大程度地减少由于不受控制的细胞旋转或翻译而模糊的图像 穿过FOV。缺少3D成像会导致对象位置的歧义，并通过焦点深度模糊 由于将3D单元投影到2D图像中。尽管在过去的几十年中，流式细胞仪系统可以 实际获取了三维（3D）空间信息的开发，与解决方案有关的限制和 样本尺寸仍然是它们最大的限制。因此，该提议的目的是开发下一个 具有高通量和高含量功能的3D成像流式细胞仪，可用于3D成像 第一次，每秒数百至数千个细胞和球状素，首次以高分辨率。我们建议 使用新型显微镜方法，线激发阵列检测显微镜开发这种细胞仪法 （LEAD），可以以当前一维细胞仪的速率在大视野中对象进行图像，但具有高3D 分辨率和高信噪比（SNR）。我们提出的铅细胞仪是一个快速扫描的灯页面 能够MHz帧速率的显微镜。我们将使用A开发最快的MHz线扫描方法 纵向示威者纵向信号驱动。我们将图像扫描的轻纸 使用线性硅光电倍数阵列，该阵列将提供如此快速扫描时需要的灵敏度， 以及如此高帧速率所需的并行读数。首先，我们将开发线性铅3D成像流 亚微米尺度分辨率和小型FOV的细胞仪。尽管我们的初步数据表明我们将 能够以100 kHz - MHz帧速率在如此高的SNR下以如此高的分辨率进行图像，我们将执行实验 测量SNR以确定铅细胞仪的工作范围。在第二个目标中，我们将增加 通过用贝塞尔束开发两光子铅成像流式细胞术来进行FOV。为了支持较大的FOV， 我们将使用八张16通道数据采集卡开发128通道数据采集系统。在第三 目的，我们将开发一个最先进的计算基础架构，该基础架构允许文件传输多达25 GB/s， 存储（> 100 TB），分析仅需成像时间3倍。我们将使用2个深度学习模型 分析。如果成功的话，这个高风险/高回报的建议将改变成像流式细胞仪局势。 提出的3D成像流式细胞仪可以在潜水员生物医学中提供改进的细胞和球形分析 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成果或诊断具有较高统计能力的患者。