Antony C. S. Chan

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.

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).

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.

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.

Subsampled scanning holographic imaging (SuSHI) for fast, non-adaptive recording of three-dimensional objects

Optical scanning holography enables the recording of three-dimensional (3D) objects involving scattering or fluorescence emission. However, the time-consuming raster scanning process prevents real-time tracking of dynamic events. We propose a compressed sensing approach to reduce the number of measurements required by scanning only along a low-density spiral trajectory, thus reducing the acquisition time. Through simulation-based performance characterization, we show that the 3D objects can be accurately restored with only 4% of holographic measurements. We also apply spiral scanning to actual holographic systems to show five to twenty-five times speed improvement in the imaging frame rate with high reconstruction fidelity. This approach thus would be critically important for single-pixel holographic recording of dynamic events, including microbead tracking and optical sectioning of 3D scenes.

All-passive pixel super-resolution of time-stretch imaging

Based on image encoding in a serial-temporal format, optical time-stretch imaging entails a stringent requirement of state-of-the-art fast data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate — hampering the widespread utilities of such technology. Here, we propose a pixel super-resolution (pixel-SR) technique tailored for time-stretch imaging that preserves pixel resolution at a relaxed sampling rate. It harnesses the subpixel shifts between image frames inherently introduced by asynchronous digital sampling of the continuous time-stretch imaging process. Precise pixel registration is thus accomplished without any active opto-mechanical subpixel-shift control or other additional hardware. Here, we present the experimental pixel-SR image reconstruction pipeline that restores high-resolution time-stretch images of microparticles and biological cells (phytoplankton) at a relaxed sampling rate (≈2–5 GSa/s)—more than four times lower than the originally required readout rate (20 GSa/s) — is thus effective for high-throughput label-free, morphology-based cellular classification down to single-cell precision. Upon integration with the high-throughput image processing technology, this pixel-SR time-stretch imaging technique represents a cost-effective and practical solution for large scale cell-based phenotypic screening in biomedical diagnosis and machine vision for quality control in manufacturing.

Reducing the Acquistion Time of Optical Scanning Holography by Compressed Sensing

We propose a compressed sensing approach to improve the the acquisition time of optical scanning holography with a spiral trajectory. A two-sectional object is reconstructed with high fidelity by at least 5 speed improvement.

Speed-dependent resolution analysis of ultrafast laser-scanning fluorescence microscopy

The image resolution of an aberration-corrected laser-scanning fluorescence microscopy (LSFM) system, like all other classical optical imaging modalities, is ultimately governed by diffraction limit and can be, in practice, influenced by the noise. However, consideration of only these two parameters is not adequate for LSFM with ultrafast laser-scanning, in which the dwell time of each resolvable image point becomes comparable with the fluorescence lifetime. In view of the continuing demand for faster LSFM, we here revisit the theoretical framework of LSFM and investigate the impact of the scanning speed on the resolution. In particular, we identify there are different speed regimes and excitation conditions in which the resolution is primarily limited by diffraction limit, fluorescence lifetime, or intrinsic noise. Our model also suggests that the speed of the current laser-scanning technologies is still at least an order of magnitude below the limit (∼sub-MHz to MHz), at which the diffraction-limited resolution can be preserved. We thus anticipate that the present study can provide new insight for practical designs and implementation of ultrafast LSFM, based on emerging laser-scanning techniques, e.g., ultrafast wavelength-swept sources, or optical time-stretch.

Pixel super-resolution in optical time-stretch microscopy using acousto-optic deflector

We present experimental demonstration of pixel super-resolution time-stretch imaging by high-speed agile-beam-steering with the use of synchronized acousto-optic deflector – enabling high-resolution imaging rate of 1MHz whereas relaxing the stringent requirement on extreme data acquisition.

Depth Enhancement of Optical Scanning Holography with a Spiral Phase Plate

A spiral phase plate is applied to the optical scanning holography system to improve the depth resolution of the reconstruction, the simulation results show that the depth interval can be resolved at a 0.4 mm with only a single hologram.