Session: 16-01-01: Government Agency Student Poster Competition

Paper Number: 149919

149919 - Light-Field Flow Cytometry: Multiparametric 3d Single-Cell Analysis With High-Throughput, High-Resolution Volumetric Multicolor Imaging 

Flow cytometry and fluorescence microscopy, known as two pivotal techniques in biomedical research, allow for rapid analysis of various cellular populations and high-resolution image acquisition of individual cells, respectively. The development of imaging flow cytometry (IFC) integrates the strengths of both techniques, providing the capabilities of high-throughput, multiparametric analysis of single cells with high spatiotemporal sensitivity and molecular specificity. Thus, the IFC technique enables the direct visualization of versatile cell properties, such as cell shape, size, biomarker intensity, and other morphological and biochemical characteristics. These advantages promise broad applications across various fundamental and translational areas, including cell biology, immunology, microbiology, hematology, and cancer research.

In recent years, significant advancements in cytometric imaging capabilities, such as speed, sensitivity, and resolution, have been achieved among the existing IFC techniques by combining different fluorescence microscopy strategies. Nevertheless, current IFC systems remain disadvantageous in higher-resolution and higher-dimensional data acquisition compared with other single-cell imaging platforms. To be specific, existing 2D IFC approaches have achieved sub-micrometer resolution with high throughput but primarily lose crucial 3D details. Alternatively, the 3D IFC techniques have been developed based on relevant microscopy techniques, such as light-sheet microscopy, confocal microscopy, beam engineering, and tomography. However, these methods may suffer from trade-offs between 3D resolution, volumetric coverage, and throughput due to sequential acquisition, as well as increased instrumental complexity and limited accessibility on commonly used microscopic platforms.

On the other hand, light-field microscopy (LFM) showcases a particularly appealing solution for capturing 3D details of fast-moving single-cell specimens by simultaneously recording the spatial-angular information of the light field. Such a scheme enables computational reconstruction of the sample volume using only a single camera frame. Moreover, recent advancements in Fourier LFM have further improved image quality and computational efficiency, facilitating 3D subcellular, millisecond spatiotemporal investigations across various biological systems ranging from functional brains, organoids to single-cell specimens. Compared to other 3D techniques, the light-field approach allows for single-shot 3D image acquisition and simple instrumentation on epi-fluorescence platforms, which are highly desirable features for cytometric imaging.

Therefore, in this work, we develop a light-field flow cytometer (LFC), a 3D IFC system designed for multiparametric 3D examination of subcellular morphology, behavior, and interactions within their native 3D contexts with high-throughput, high-resolution volumetric multicolor imaging. The LFC system is developed on a high-resolution light-field optofluidic platform with hydrodynamic focusing and stroboscopic illumination. It provides a throughput of more than 5,000 particles per second, a near-diffraction-limited resolution, and the multi-color capability for analyzing 3D subcellular structures, overcoming the tradeoff between resolution and throughput in IFC while maintaining the unique 3D snapshot capability of light-field imaging. We demonstrate the system by interrogating and quantifying a wide range of phantoms and biological morphologies, functions, and heterogeneities. These include imaging of peroxisomes and mitochondria in cultured cells, morphological characterizations of isolated cells from mice and humans, apoptotic alterations in staurosporine-treated Jurkat cells, and the expression of tdTomato following Cre mRNA delivery in cells from different mouse organs. Notably, this system features low instrumental complexity, making it compatible with commonly used epi-fluorescence microscopes and microfluidic devices. Hence, we anticipate that LFC, as a readily accessible and compatible cytometric imaging technique, will significantly advance a wide range of cytometric imaging applications across various fields such as biology, pharmacology, and medical diagnostics.

Presenting Author: Xuanwen Hua Georgia Institute of Technology

Presenting Author Biography: Dr. Xuanwen Hua earned his Bachelor of Science degree in applied physics from the University of Science and Technology of China in 2016. Xuanwen completed his Ph.D. in biomedical engineering from the Georgia Institute of Technology and Emory University in May 2024 and began his postdoctoral position in April 2024. Xuanwen has more than six years of research experience in optical microscopy, focusing on optical wavefront modulation, super-resolution microscopy, light-field microscopy, imaging flow cytometry, and deep learning. His research work has been published in several top-level journals, including Optica, Science Advances, and Nature Communications.

Authors:

Xuanwen Hua Georgia Institute of Technology
Keyi Han Georgia Institute of Technology
Shu Jia Georgia Institute of Technology