Anson H. L. Tang

Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

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.

Ultrafast laser-scanning time-stretch imaging at visible wavelengths

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.

High-throughput time-stretch imaging flow cytometry for multi-class classification of phytoplankton

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.

Optical Time Stretch for High-Speed and High-Throughput Imaging—From Single-Cell to Tissue-Wide Scales

Initially developed for high-speed optical communication, optical time stretch has recently been adopted for ultrafast and sensitive optical imaging at an unprecedented speed. In this paper, we highlight the essential concepts as well as the enabling elements of this ultrafast technology. More importantly, we review the recent developments of optical time-stretch imaging, especially in the context of 1) quantitative optofluidic microscopy for high-content single-cell phenotyping at an imaging throughput ~100 000 cells/s; 2) all-optical multi-MHz (>10 MHz) swept-source optical coherence tomography (OCT) for high-speed in vivo anatomical and functional 3-D tissue imaging. We also discuss the current technological challenges in time-stretch imaging. In particular, generating the enormous data in real time, this technology could uniquely create new insights of data-driven science in clinical diagnostics and basic biological research.

Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay

Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

Multi-MHz laser-scanning single-cell fluorescence microscopy by spatiotemporally encoded virtual source array

Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Ultrawide C- and L-band mode-locked erbium-doped fiber ring laser and its application in ultrafast microscopy

We report an ultrawide C- and L-band erbium-doped fiber ring laser with 92-nm bandwidth centered at 1550 nm and 44.5-MHz repetition rate. Furthermore, it was applied to ultrafast time-stretch microscopy.

Optical time-stretch microscopy enabled by free-space angular-chirp-enhanced delay (Conference Presentation)

Optical time-stretch microscopy enables cellular images captured at tens of MHz line-scan rate and becomes a potential tool for ultrafast dynamics monitoring and high throughput screening in scientific and biomedical applications. In time-stretch microscopy, to achieve the fast line-scan rate, optical fibers are used as the pulse-stretching device that maps the spectrum of a light pulse to a temporal waveform for fast digitization. Consequently, existing time-stretch microscopy is limited to work at telecom windows (e.g. 1550 nm) where optical fiber has significant pulse-stretching and small loss. This limitation circumscribes the potential application of time-stretch microscopy. Here we present a new optical time-stretch imaging modality by exploiting a novel pulse-stretching technique, free-space angular-chirp-enhanced delay (FACED), which has three benefits: (1) Pulse-stretching in FACED generates substantial, reconfigurable temporal dispersion in free-space with low intrinsic loss at visible wavelengths; (2) Pulse-stretching in FACED inherently provides an ultrafast all-optical laser-beam scanning mechanism for time-stretch imaging. (3) Pulse-stretching in FACED can be wavelength-invariant, which enables time-stretch microscopy implemented without spectral-encoding. Using FACED, we demonstrate optical time-stretch microscopy with visible light (~700 nm). Compared to the prior work, bright-field time-stretch images captured show superior contrast and resolution, and can be effectively colorized to generate color time-stretch images. More prominently, accessing the visible spectrum regime, we demonstrate that FACED enables ultrafast fluorescence time-stretch microscopy. Our results suggest FACED could unleash a wider scope of applications that were once forbidden with the fiber based time-stretch imaging techniques.

Ultrafast quantitative time-stretch imaging flow cytometry of phytoplankton

Comprehensive quantification of phytoplankton abundance, sizes and other parameters, e.g. biomasses, has been an important, yet daunting task in aquatic sciences and biofuel research. It is primarily because of the lack of effective tool to image and thus accurately profile individual microalgae in a large population. The phytoplankton species are highly diversified and heterogeneous in terms of their sizes and the richness in morphological complexity. This fact makes time-stretch imaging, a new ultrafast real-time optical imaging technology, particularly suitable for ultralarge-scale taxonomic classification of phytoplankton together with quantitative image recognition and analysis. We here demonstrate quantitative imaging flow cytometry of single phytoplankton based on quantitative asymmetric-detection time-stretch optical microscopy (Q-ATOM) – a new time-stretch imaging modality for label-free quantitative phase imaging without interferometric implementations. Sharing the similar concept of Schlieren imaging, Q-ATOM accesses multiple phase-gradient contrasts of each single phytoplankton, from which the quantitative phase profile is computed. We employ such system to capture, at an imaging line-scan rate of 11.6 MHz, high-resolution images of two phytoplankton populations (scenedesmus and chlamydomonas) in ultrafast microfluidic flow (3 m/s). We further perform quantitative taxonomic screening analysis enabled by this technique. More importantly, the system can also generate quantitative phase images of single phytoplankton. This is especially useful for label-free quantification of biomasses (e.g. lipid droplets) of the particular species of interest – an important task adopted in biofuel applications. Combining machine learning for automated classification, Q-ATOM could be an attractive platform for continuous and real-time ultralarge-scale single-phytoplankton analysis.