Optical time-stretch imaging enables the continuous capture of non-repetitive events in real time at a line-scan rate of tens of MHz—a distinct advantage for the ultrafast dynamics monitoring and high-throughput screening that are widely needed in biological microscopy. However, its potential is limited by the technical challenge of achieving significant pulse stretching (that is, high temporal dispersion) and low optical loss, which are the critical factors influencing imaging quality, in the visible spectrum demanded in many of these applications. We present a new pulse-stretching technique, termed free-space angular-chirp-enhanced delay (FACED), with three distinguishing features absent in the prevailing dispersive-fiber-based implementations: (1) it generates substantial, reconfigurable temporal dispersion in free space (>1 ns nm−1) with low intrinsic loss (<6 dB) at visible wavelengths; (2) its wavelength-invariant pulse-stretching operation introduces a new paradigm in time-stretch imaging, which can now be implemented both with and without spectral encoding; and (3) pulse stretching in FACED inherently provides an ultrafast all-optical laser-beam scanning mechanism at a line-scan rate of tens of MHz. Using FACED, we demonstrate not only ultrafast laser-scanning time-stretch imaging with superior bright-field image quality compared with previous work but also, for the first time, MHz fluorescence and colorized time-stretch microscopy. Our results show that this technique could enable a wider scope of applications in high-speed and high-throughput biological microscopy that were once out of reach.

Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay.

A growing body of evidence has substantiated the significance of quantitative phase imaging (QPI) in enabling cost‐effective and label‐free cellular assays, which provides useful insights into understanding the 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 cells—hindering their utility in mainstream biomedicine and biology. Here we present a new QPI modality, coined multiplexed asymmetric‐detection time‐stretch optical microscopy (multi‐ATOM) that captures and processes quantitative label‐free single‐cell images at ultrahigh throughput without compromising subcellular 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, multivariate, cell‐type classification (e.g. breast cancer subtypes, and leukemic cells vs 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.

Time-stretch imaging has been regarded as an attractive technique for high-throughput imaging flow cytometry primarily owing to its real-time, continuous ultrafast operation. Nevertheless, two key challenges remain: (1) sufficiently high time-stretch image resolution and contrast is needed for visualizing sub-cellular complexity of single cells, and (2) the ability to unravel the heterogeneity and complexity of the highly diverse population of cells - a central problem of single-cell analysis in life sciences - is required. We here demonstrate an optofluidic time-stretch imaging flow cytometer that enables these two features, in the context of high-throughput multi-class (up to 14 classes) phytoplantkton screening and classification. Based on the comprehensive feature extraction and selection procedures, we show that the intracellular texture/morphology, which is revealed by high-resolution time-stretch imaging, plays a critical role of improving the accuracy of phytoplankton classification, as high as 94.7%, based on multi-class support vector machine (SVM). We also demonstrate that high-resolution time-stretch images, which allows exploitation of various feature domains, e.g. Fourier space, enables further sub-population identification - paving the way toward deeper learning and classification based on large-scale single-cell images. Not only applicable to biomedical diagnostic, this work is anticipated to find immediate applications in marine and biofuel research.

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