Broadband fiber-optical parametric amplification for ultrafast time-stretch imaging at 1.0 µm.

We demonstrate a broadband all-fiber-optical parametric amplifier for ultrafast time-stretch imaging at 1.0 µm, featured by its compact design, alignment-free, high efficiency, and flexible gain spectrum through fiber nonlinearity- and dispersion-engineering: specifically on a dispersion-stabilized photonic-crystal fiber (PCF) to achieve a net gain over 20 THz (75 nm) and a highest gain of ~6000 (37.5 dB). Another unique feature of the parametric amplifier, over other optical amplifiers, is the coherent generation of a synchronized signal replica (called idler) that can be exploited to offer an extra 3-dB gain by optically superposing the signal and idler. It further enhances signal contrast and temporal stability. For proof-of-concept purpose, ultrahigh speed and diffraction-limited time-stretch microscopy is demonstrated with a single-shot line-scan rate of 13 MHz based on the dual-band (signal and idler) detection. Our scheme can be extended to other established bioimaging modalities, such as optical coherence tomography, near infrared fluorescence, and photoacoustic imaging, where weak signal detection at high speed is required.

Breathing laser as an inertia-free swept source for high-quality ultrafast optical bioimaging.

We demonstrate an all-fiber breathing laser as inertia-free swept source (BLISS), with an ultra-compact design, for the emerging ultrafast bioimaging modalities. The unique feature of BLISS is its broadband wavelength-swept operation (∼60 nm) with superior temporal stability in terms of both long term (0.08 dB over 27 h) and shot-to-shot power variations (2.1%). More importantly, it enables a wavelength sweep rate of >10 MHz (∼7×108 nm/s)­orders-of-magnitude faster than the existing swept sources based on mechanical or electrical tuning techniques. BLISS thus represents a practical and new generation of swept source operating in the unmet megahertz swept-rate regime that aligns with the pressing need for scaling the optical bioimaging speed in ultrafast phenomena study or high-throughput screening applications. To showcase its utility in high-speed optical bioimaging, we here employ BLISS for ultrafast time-stretch microscopy and multi-MHz optical coherence tomography of the biological specimen at a single-shot line-scan rate or A-scan rate of 11.5 MHz.

Optical time-stretch confocal microscopy at 1 µm.

We demonstrate optical time-stretch confocal microscopy in the 1 µm spectral window for high-speed and high-resolution cellular imaging. In contrast to the prior demonstrations of time-stretch imaging, which all operated in the telecommunication band, the present work extends the utility of this imaging modality to a wavelength regime (~1 µm), which is well known to be the optimal diagnostic window in biophotonics. This imaging technique enables us to image the nasopharyngeal epithelial cells with cellular resolution (<2 µm), at a line scan rate of 10 MHz, and with a field of view as wide as ~0.44 mm × 0.1 mm. We also theoretically and experimentally characterized the system performance. As the low-loss dispersive fibers for the time-stretch process as well as other essential optical components for enhancing the imaging sensitivity are commonly available at 1 µm, time-stretch confocal microscopy in this wavelength range could usher in realizing high-speed cell imaging with an unprecedented throughput.

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

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.

Correction: Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry.

Correction for 'Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry' by Andy K. S. Lau et al., Lab Chip, 2016, 16, 1743-1756.

Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry.

Optical imaging is arguably the most effective tool to visualize living cells with high spatiotemporal resolution and in a nearly noninvasive manner. Driven by this capability, state-of-the-art cellular assay techniques have increasingly been adopting optical imaging for classifying different cell types/stages, and thus dissecting the respective cellular functions. However, it is still a daunting task to image and characterize cell-to-cell variability within an enormous and heterogeneous population - an unmet need in single-cell analysis, which is now widely advocated in modern biology and clinical diagnostics. The challenge stems from the fact that current optical imaging technologies still lack the practical speed and sensitivity for measuring thousands to millions of cells down to the single-cell precision. Adopting the wisdom in high-speed fiber-optics communication, optical time-stretch imaging has emerged as a completely new optical imaging concept which is now proven for ultrahigh-throughput optofluidic single-cell imaging, at least 1-2 orders-of-magnitude higher (up to ∼100 000 cells per second) compared to the existing imaging flow cytometers. It also uniquely enables quantification of intrinsic biophysical markers of individual cells - a largely unexploited class of single-cell signatures that is known to be correlated with the overwhelmingly investigated biochemical markers. With the aim of reaching a wider spectrum of experts specializing in cellular assay developments and applications, this paper highlights the essential basics of optical time-stretch imaging, followed by reviewing the recent developments and applications of optofluidic time-stretch imaging. We will also discuss the current challenges of this technology, in terms of providing new insights in basic biology and enriching the clinical diagnostic toolsets.

Ultrafast Microfluidic Cellular Imaging by Optical Time-Stretch.

There is an unmet need in biomedicine for measuring a multitude of parameters of individual cells (i.e., high content) in a large population efficiently (i.e., high throughput). This is particularly driven by the emerging interest in bringing Big-Data analysis into this arena, encompassing pathology, drug discovery, rare cancer cell detection, emulsion microdroplet assays, to name a few. This momentum is particularly evident in recent advancements in flow cytometry. They include scaling of the number of measurable colors from the labeled cells and incorporation of imaging capability to access the morphological information of the cells. However, an unspoken predicament appears in the current technologies: higher content comes at the expense of lower throughput, and vice versa. For example, accessing additional spatial information of individual cells, imaging flow cytometers only achieve an imaging throughput ~1000 cells/s, orders of magnitude slower than the non-imaging flow cytometers. In this chapter, we introduce an entirely new imaging platform, namely optical time-stretch microscopy, for ultrahigh speed and high contrast label-free single-cell (in a ultrafast microfluidic flow up to 10 m/s) imaging and analysis with an ultra-fast imaging line-scan rate as high as tens of MHz. Based on this technique, not only morphological information of the individual cells can be obtained in an ultrafast manner, quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase is also possible. The technology can also be integrated with conventional fluorescence measurements widely adopted in the non-imaging flow cytometers. Therefore, these two combinatorial and complementary measurement capabilities in long run is an attractive platform for addressing the pressing need for expanding the "parameter space" in high-throughput single-cell analysis. This chapter provides the general guidelines of constructing the optical system for time stretch imaging, fabrication and design of the microfluidic chip for ultrafast fluidic flow, as well as the image acquisition and processing.

28 MHz swept source at 1.0 µm for ultrafast quantitative phase imaging.

Emerging high-throughput optical imaging modalities, in particular those providing phase information, necessitate a demanding speed regime (e.g. megahertz sweep rate) for those conventional swept sources; while an effective solution is yet to be demonstrated. We demonstrate a stable breathing laser as inertia-free swept source (BLISS) operating at a wavelength sweep rate of 28 MHz, particularly for the ultrafast interferometric imaging modality at 1.0 µm. Leveraging a tunable dispersion compensation element inside the laser cavity, the wavelength sweep range of BLISS can be tuned from ~10 nm to ~63 nm. It exhibits a good intensity stability, which is quantified by the ratio of standard deviation to the mean of the pulse intensity, i.e. 1.6%. Its excellent wavelength repeatability, <0.05% per sweep, enables the single-shot imaging at an ultrafast line-scan rate without averaging. To showcase its potential applications, it is applied to the ultrafast (28-MHz line-scan rate) interferometric time-stretch (iTS) microscope to provide quantitative morphological information on a biological specimen at a lateral resolution of 1.2 µm. This fiber-based inertia-free swept source is demonstrated to be robust and broadband, and can be applied to other established imaging modalities, such as optical coherence tomography (OCT), of which an axial resolution better than 12 µm can be achieved.

Interferometric time-stretch microscopy for ultrafast quantitative cellular and tissue imaging at 1 µm.

Quantitative phase imaging (QPI) has been proven to be a powerful tool for label-free characterization of biological specimens. However, the imaging speed, largely limited by the image sensor technology, impedes its utility in applications where high-throughput screening and efficient big-data analysis are mandated. We here demonstrate interferometric time-stretch (iTS) microscopy for delivering ultrafast quantitative phase cellular and tissue imaging at an imaging line-scan rate >20 MHz­orders-of-magnitude faster than conventional QPI. Enabling an efficient time-stretch operation in the 1-µm wavelength window, we present an iTS microscope system for practical ultrafast QPI of fixed cells and tissue sections, as well as ultrafast flowing cells (at a flow speed of up to 8 m∕s). To the best of our knowledge, this is the first time that time-stretch imaging could reveal quantitative morphological information of cells and tissues with nanometer precision. As many parameters can be further extracted from the phase and can serve as the intrinsic biomarkers for disease diagnosis, iTS microscopy could find its niche in high-throughput and high-content cellular assays (e.g., imaging flow cytometry) as well as tissue refractometric imaging (e.g., whole-slide imaging for digital pathology).

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.