Tang Anson H L, Lai Queenie T K, Chung Bob M F, Lee Kelvin C M, Mok Aaron T Y, Yip G K, Shum Anderson H C, Wong Kenneth K Y, Tsia Kevin K
Department of Electrical and Electronic Engineering, The University of Hong Kong.
Department of Mechanical Engineering, The University of Hong Kong.
J Vis Exp. 2017 Jun 28(124):55840. doi: 10.3791/55840.
Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.
扩大可测量参数的数量，这使得多维度数据分析成为可能，从而获得更高可信度的统计结果，一直是流式细胞术先进发展的主要趋势。值得注意的是，增加高分辨率成像能力可对细胞/亚细胞结构进行复杂的形态分析。这对于标准流式细胞仪来说是不可能的。然而，这对于推进我们对细胞功能的认识很有价值，并且可以使生命科学研究、临床诊断和环境监测受益。将成像能力整合到流式细胞术中会影响检测通量，主要是由于相机技术在速度和灵敏度方面的限制。为了在保持图像质量的同时克服成像流式细胞术面临的速度或通量挑战，非对称检测时间拉伸光学显微镜（ATOM）已被证明能够以高达100,000个细胞/秒的成像通量实现具有亚细胞分辨率的高对比度单细胞成像。基于传统时间拉伸成像的成像概念，即通过使用超快宽带激光脉冲进行全光图像编码和检索，ATOM通过增强未标记/未染色细胞的图像对比度进一步提升成像性能。这是通过获取细胞的相位梯度信息来实现的，该信息被光谱编码到单次宽带脉冲中。因此，ATOM在单细胞形态和纹理的高通量测量中特别有利，这些信息可指示细胞类型、状态甚至功能。最终，这可能成为一个强大的用于细胞生物物理表型分析的成像流式细胞术平台，补充当前基于生化标记的细胞检测的技术水平。这项工作描述了一个协议，以建立ATOM系统的关键模块（从光学前端到数据处理和可视化后端），以及基于ATOM的成像流式细胞术的工作流程，以人类细胞和微藻为例。
