Multi-ATOM: Ultrahigh-throughput single-cell quantitative phase imaging with subcellular resolution

Abstract

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost-effective and label-free cellular assay, which provides useful insights into understanding biophysical properties of cells and their roles in cellular functions. However, available QPI modalities are limited by the loss of imaging resolution at high throughput and thus run short of sufficient statistical power at the single cell precision to define cell identities in a large and heterogeneous population of cellshindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multi-ATOM that captures and processes quantitative label-free single-cell images at ultra-high throughput without compromising sub-cellular resolution. We show that multi-ATOM, based upon ultrafast phase-gradient encoding, outperforms state-of-the-art QPI in permitting robust phase retrieval at a QPI throughput of >10,000 cell/sec, bypassing the need for interferometry which inevitably compromises QPI quality under ultrafast operation. We employ multi-ATOM for large-scale, label-free, multi-variate, cell-type classification (e.g. breast cancer sub-types, and leukemic cells versus peripheral blood mononuclear cells) at high accuracy (>94%). Our results suggest that multi-ATOM could empower new strategies in large-scale biophysical single-cell analysis with applications in biology and enriching disease diagnostics.

Quantitative Phase Imaging Flow Cytometry for Ultra‐Large‐Scale Single‐Cell Biophysical Phenotyping

“…The detailed working principle can be referred to Ref. . In brief, multi‐ATOM effectively measures the local phase gradient induced by the imaged cells—resulting in intensity variation that can be measured through partial beam‐block on the Fourier plane of the imaged cells (i.e., the multi‐ATOM module in Fig.…”

“…In contrast to the vast majority of the current QPI modalities, multi‐ATOM requires no interferometry and no computationally intensive iterative method for phase retrieval . Combining with high‐performance computing, these important attributes enables robust continuous high‐throughput, blur‐free QPI without losing image resolution (Fig.…”

“…Both the magnitude and direction of the 2D local phase gradient, and thus quantitative phase, are retrieved based on an optical module based on a simple assembly of free‐space and fiber optics components [i.e., multi‐ATOM module in Fig. a ] . In this module, a one‐to‐four de‐multiplexer splits the image‐encoded pulsed beam into four identical replicas, each of which is half‐blocked by a knife‐edge from four different orientations (right, left, top, and bottom).…”

“…Here we present an ultra‐large‐scale single‐cell biophysical phenotyping approach through a newly developed QPI platform, coined multiplexed asymmetric‐detection time‐stretch optical microscopy [ multi‐ATOM ] , to address the aforementioned challenges. This technique features with ultrafast QPI encoding that overcomes the speed limitation of classic QPI as well as the loss (and thus resolution) limitation of interferometric time‐stretch QPI.…”

A review of microscopic cell imaging and neural network recognition for synergistic cyanobacteria identification and enumeration

“…Integration of QPI units with flow cytometry equipment provides capabilities for enhanced analysis relative to conventional imaging flow cytometry techniques (Table 2). Combined flow cytometry QPI devices process samples at rates considerably higher than those achievable through manual analysis, expediating specimen identification and providing larger data sets for neural network training [20,35,37]. In addition to capturing high resolution images, additional biophysical parameters not accessible through conventional imaging flow cytometry are made available, including those previously outlined [37].…”