无标记成像流式细胞术是生物和医学研究的强大工具,因为它克服了主要依赖于荧光标记的常规基于荧光的成像流式细胞术的技术挑战。迄今为止,已经开发了两种不同类型的无标记成像流式细胞术,即光流体时间拉伸定量相位成像流式细胞术和受激拉曼散射(SRS)成像流式细胞术。不幸的是,这两种方法无法探测到一些重要的分子,如淀粉和胶原蛋白。这里,我们提出了另一种类型的无标记成像流式细胞术,即多光子成像流式细胞术,用于高通量可视化活细胞中的淀粉和胶原蛋白。我们的多光子成像流式细胞仪基于非线性光学成像,其图像对比度由两种光学非线性效应提供:四波混合(FWM)和二次谐波产生(SHG)。它由带有声学聚焦器的微流控芯片组成,实验室制造的激光扫描SHG-FWM显微镜,和高速图像采集电路,以同时采集流动细胞的FWM和SHG图像。因此,它以每秒四到五个事件的高事件速率,以500nm的空间分辨率和50μm×50μm的视场获取FWM和SHG图像(100×100像素),对应于560-700kb/s的高吞吐量,其中事件由细胞或细胞样颗粒的通过定义。为了展示我们的多光子成像流式细胞仪的实用性,我们用它来表征绿虫(NIES-2175),一种单细胞绿藻,最近吸引了工业部门的注意,因为它能够有效地生产生物塑料的有价值的材料,食物,和生物燃料。我们的统计图像分析发现,淀粉在细胞周期早期阶段分布在细胞中心,并在后期变得离域。多光子成像流式细胞术有望成为统计高含量生物功能研究和优化高产细胞株进化的有效工具。
Label-free imaging flow cytometry is a powerful tool for biological and medical research as it overcomes technical challenges in conventional fluorescence-based imaging flow cytometry that predominantly relies on fluorescent labeling. To date, two distinct types of label-free imaging flow cytometry have been developed, namely optofluidic time-stretch quantitative phase imaging flow cytometry and stimulated Raman scattering (SRS) imaging flow cytometry. Unfortunately, these two methods are incapable of probing some important molecules such as starch and collagen. Here, we present another type of label-free imaging flow cytometry, namely multiphoton imaging flow cytometry, for visualizing starch and collagen in live cells with high throughput. Our multiphoton imaging flow cytometer is based on nonlinear optical imaging whose image contrast is provided by two optical nonlinear effects: four-wave mixing (FWM) and second-harmonic generation (SHG). It is composed of a microfluidic chip with an acoustic focuser, a lab-made laser scanning SHG-FWM microscope, and a high-speed image acquisition circuit to simultaneously acquire FWM and SHG images of flowing cells. As a result, it acquires FWM and SHG images (100 × 100 pixels) with a spatial resolution of 500 nm and a field of view of 50 μm × 50 μm at a high event rate of four to five events per second, corresponding to a high throughput of 560-700 kb/s, where the event is defined by the passage of a cell or a cell-like particle. To show the utility of our multiphoton imaging flow cytometer, we used it to characterize Chromochloris zofingiensis (NIES-2175), a unicellular green alga that has recently attracted attention from the industrial sector for its ability to efficiently produce valuable materials for bioplastics, food, and biofuel. Our statistical image analysis found that starch was distributed at the center of the cells at the early cell cycle stage and became delocalized at the later stage. Multiphoton imaging flow cytometry is expected to be an effective tool for statistical high-content studies of biological functions and optimizing the evolution of highly productive cell strains.