Andy K. S. Lau

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.

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.

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

Abstract. 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  MHz—orders-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).

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.

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.

Coherent Laser Source for High Frame-Rate Optical Time-Stretch Microscopy at 1.0 μm

We demonstrate a coherent picosecond pulsed fiber laser for the high frame-rate optical time-stretch microscopy at 1.0 μm. The spectrum of a picosecond pulsed laser is commonly broadened before the time-stretch imaging, which however will degrade its stability and coherence. As a result, it is required to enhance the degraded signal-to-noise ratio by averaging, which would compromise the frame rate on the other hand. Instead of pursuing such kind of spectrum-broadened picosecond pulsed laser sources, we propose a pulse train extracted directly from an all-normal dispersion mode-locked fiber laser with a rectangle-shaped optical spectrum. It delivers stable and coherent performance for the serial time-encoded amplified microscopy at 1.0 μm. With this robust picosecond pulsed laser, real-time stain-free flow imaging with a frame rate of 26.25 MHz and a spatial resolution of <; 2 μm is demonstrated. Featured with the compact configuration and good coherence property, it is a promising picosecond pulsed fiber laser source for the ultrafast interferometric time-stretch microscopy at 1.0 μm.

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.

Arbitrary two-dimensional spectrally encoded pattern generation—a new strategy for high-speed patterned illumination imaging

Patterned illumination is now a proven technique in optical metrology and imaging, and is widely employed in both industrial and biological applications. However, existing techniques are unable to meet the pressing need for higher imaging rates. To address this challenge, we propose and demonstrate two-dimensional (2D) mechanical-scan-free arbitrary patterned illumination by 2D spectral encoding. The illumination pattern is flexibly generated at high speed by spectral shaping together with wavelength-to-2D space mapping. Performance optimization of this new patterned illumination scheme (e.g., pattern distortion and resolution) is generally an ill-defined problem involving multiple interrelated parameters. We demonstrate proof-of-principle experiments based on a multiobjective optimization routine using a genetic algorithm to validate our optimization model as well as to show the feasibility of patterned illumination by use of spectral interferometry. Adopting the wisdom of high-speed arbitrary waveform generation routinely practiced in telecommunications as well as ultrafast wavelength-sweep mechanisms, the proposed method could have an impact on advanced imaging modalities, particularly when speed is of a critical concern.

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.

Accelerated cell imaging and classification on FPGAs for quantitative-phase asymmetric-detection time-stretch optical microscopy

With the fundamental trade-off between speed and sensitivity, existing quantitative phase imaging (QPI) systems for diagnostics and cell classification are often limited to batch processing only small amount of offline data. While quantitative asymmetric-detection time-stretch optical microscopy (Q-ATOM) offers a unique optical platform for ultrafast and high-sensitivity quantitative phase cellular imaging, performing the computationally demanding backend QPI phase retrieval and image classification in real-time remains a major technical challenge. In this paper, we propose an optimized architecture for QPI on FPGA and compare its performance against CPU and GPU implementations in terms of speed and power efficiency. Results show that our implementation on single FPGA card demonstrates a speedup of 9.4 times over an optimized C implementation running on a 6-core CPU, and 3.47 times over the GPU implementation. It is also 24.19 and 4.88 times more power-efficient than the CPU and GPU implementation respectively. Throughput increase linearly when four FPGA cards are used to further improve the performance. We also demonstrate an increased classification accuracy when phase images instead of single-angle ATOM images are used. Overall, one FPGA card is able to process and categorize 2497 cellular images per second, making it suitable for real-time single-cell analysis applications.