Single-molecule fluorescence-activated cell sorting (smFACS): sorting cells molecule by molecule

本项目的目标是开发具有单分子灵敏度的流式细胞仪，用于表征和分选具有低拷贝数膜蛋白的细胞。由于细胞产生的背景荧光远大于单个荧光抗体产生的发射，当代流式细胞仪无法区分单个分子。这将通过开发一种新的灵敏成像流式细胞仪设备来克服，该设备将产生每个细胞的3D表示以可视化单个膜蛋白。这种微流控装置将使用高速单分子光片显微镜，克服了当代细胞仪的灵敏度限制，能够以高流速（高达100个细胞/秒）表征细胞。由于高图像采集速率（100 kHz），因此有必要在嵌入式成像平台上对图像进行真实的实时分析。现场可编程门阵列（FPGA）将用于高速相机接口，数据流和外围设备接口，而图形处理单元将用于图像处理。定义良好的解决方案集适用于AI/深度学习解决方案，预计该系统的处理速度将超过传统方法三个数量级。这不仅将确保流式细胞术的高通量速率，而且还将实现具有单分子灵敏度（smFACS）的荧光激活细胞分选（FACS）。开发的设备将用于探索如何提高灵敏度可以帮助筛选患者细胞。该项目将推动单分子成像的速度限制，最终目标是在医院中使用这种设备，以最大限度地增加可以通过靶向细胞膜上表达的特定分子进行治疗的患者数量。

The goal of this project is to develop a flow cytometer with single-molecule sensitivity, for characterising and sorting cells with low copy numbers of membrane proteins. Contemporary flow cytometers cannot distinguish individual molecules due to the background fluorescence produced by cells, which is much larger than the emission produced by a single fluorescent antibody. This will be overcome by developing a novel sensitive imaging flow cytometry device, that will produce a 3D representation of each cell to visualise individual membrane proteins. This microfluidic device will use high-speed single-molecule light-sheet microscopy, which overcomes sensitivity limitations of contemporary cytometers to enable characterisation of cells at high flow rates (up to 100 cells/s). Because of the high image acquisition rate (100 kHz), it will be necessary to analyse images in real time on an embedded imaging platform. Field Programmable Gate Arrays (FPGA) will be utilised for the high-speed camera interface, data flow and peripheral interface, while graphics processing units will be employed for image processing. The well-defined solution set lends itself to AI/deep learning solutions and it is expected that this system will have a processing rate of three orders of magnitude beyond traditional approaches. Not only will this ensure a high throughput rate for flow cytometry, but it will also enable fluorescence-activated cell sorting (FACS) with single-molecule sensitivity (smFACS). Developed devices will be used to explore how the increased sensitivity can aid in screening patient cells. This project will push the limits of how fast single-molecule imaging can be performed and ultimately the goal is for such a device to be employed in hospitals to maximise the number of patients that can be treated by targeting specific molecules expressed on cell membranes.