Multimodal FACED imaging for large-scale single-cell morphological profiling

Gwinky G K Yip,Wenwei Yan,Kevin K Tsia,Kelvin C M Lee,Kenneth K Y Wong,Queenie T K Lai,Michelle C K Lo

doi:10.1063/5.0054714

Gwinky G K Yip, Wenwei Yan + Show 5 more

Open Access

https://doi.org/10.1063/5.0054714

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Abstract

Free-space angular-chirp-enhanced delay (FACED) is an ultrafast laser-scanning technique that allows for high imaging speed at the scale orders of magnitude greater than the current technologies. However, this speed advantage has only been restricted to bright-field and fluorescence imaging—limiting the variety of image contents and hindering its applicability in image-based bioassay, which increasingly demands rich phenotypic readout at a large scale. Here, we present a new high-speed quantitative phase imaging (QPI) based on time-interleaved phase-gradient FACED image detection. We further integrate this system with a microfluidic flow cytometer platform that enables synchronized and co-registered single-cell QPI and fluorescence imaging at an imaging throughput of 77 000 cells/s with sub-cellular resolution. Combined with deep learning, this platform empowers comprehensive image-based profiling of single-cell biophysical phenotypes that can offer not only sufficient label-free power for cell-type classification but also cell-cycle phase tracking with high accuracy comparable to the gold-standard fluorescence method. This platform further enables correlative, compartment-specific single-cell analysis of the spatially resolved biophysical profiles at the throughput inaccessible with existing QPI methods. The high imaging throughput and content given by this multimodal FACED imaging system could open new opportunities in image-based single-cell analysis, especially systematic analysis that correlates the biophysical and biochemical information of cells, and provide new mechanistic insights into biophysical heterogeneities in many biological processes.

Highlights

Recent advances in optical microscopy have opened an increasingly detailed window into visualizing and understanding how biological cells—the basic unit of biological systems—work and fail
Note that this imaging throughput is at least two orders of magnitude higher than the existing quantitative phase imaging (QPI) systems that combine with fluorescence imaging capability
By leveraging an all-optical passive laser scanner based on Free-space angular-chirp-enhanced delay (FACED), we have demonstrated a high-throughput QPI modality that achieves an ultrafast imaging line-scan rate beyond MHz and preserves the subcellular resolution

Summary

Introduction

Recent advances in optical microscopy have opened an increasingly detailed window into visualizing and understanding how biological cells—the basic unit of biological systems—work and fail. Several approaches further incorporated fluorescence imaging with 2D (or even 3D) QPI to enable simultaneous readout of both molecular information specific to the subcellular organelles/molecules (given by fluorescence contrast) scitation.org/journal/app and biophysical information (derived from QPI).. Several approaches further incorporated fluorescence imaging with 2D (or even 3D) QPI to enable simultaneous readout of both molecular information specific to the subcellular organelles/molecules (given by fluorescence contrast) scitation.org/journal/app and biophysical information (derived from QPI).8–12 It remains restricted in adopting these integrated QPI systems in the growing field of image-based profiling. It is mainly because these platforms lack the imaging throughput (e.g., in the number of cells per second) required to offer sufficient statistical power and establish faithful correlation and/or validation of biophysical phenotypes with the foundational molecular knowledge

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