Terence T. W. Wong

High-speed label-free functional photoacoustic microscopy of mouse brain in action

We present fast functional photoacoustic microscopy (PAM) for three-dimensional high-resolution, high-speed imaging of the mouse brain, complementary to other imaging modalities. We implemented a single-wavelength pulse-width-based method with a one-dimensional imaging rate of 100 kHz to image blood oxygenation with capillary-level resolution. We applied PAM to image the vascular morphology, blood oxygenation, blood flow and oxygen metabolism in both resting and stimulated states in the mouse brain.

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).

In vivo deep brain imaging of rats using oral-cavity illuminated photoacoustic computed tomography

Abstract. Using internal illumination with an optical fiber in the oral cavity, we demonstrate, for the first time, photoacoustic computed tomography (PACT) of the deep brain of rats in vivo. The experiment was performed on a full-ring-array PACT system, with the capability of providing high-speed cross-sectional imaging of the brain. Compared with external illumination through the cranial skull, internal illumination delivers more light to the base of the brain. Consequently, in vivo photoacoustic images clearly reveal deep brain structures such as the hypothalamus, brain stem, and cerebral medulla.

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.

Fast label-free multilayered histology-like imaging of human breast cancer by photoacoustic microscopy

A photoacoustic microscope system provides label-free multilayered histology-like imaging of unprocessed human breast specimens. The goal of breast-conserving surgery is to completely remove all of the cancer. Currently, no intraoperative tools can microscopically analyze the entire lumpectomy specimen, which results in 20 to 60% of patients undergoing second surgeries to achieve clear margins. To address this critical need, we have laid the foundation for the development of a device that could allow accurate intraoperative margin assessment. We demonstrate that by taking advantage of the intrinsic optical contrast of breast tissue, photoacoustic microscopy (PAM) can achieve multilayered histology-like imaging of the tissue surface. The high correlation of the PAM images to the conventional histologic images allows rapid computations of diagnostic features such as nuclear size and packing density, potentially identifying small clusters of cancer cells. Because PAM does not require tissue processing or staining, it can be performed promptly and intraoperatively, enabling immediate directed re-excision and reducing the number of second surgeries.

Label-free automated three-dimensional imaging of whole organs by microtomy-assisted photoacoustic microscopy

Three-dimensional (3D) optical imaging of whole biological organs with microscopic resolution has remained a challenge. Most versions of such imaging techniques require special preparation of the tissue specimen. Here we demonstrate microtomy-assisted photoacoustic microscopy (mPAM) of mouse brains and other organs, which automatically acquires serial distortion-free and registration-free images with endogenous absorption contrasts. Without tissue staining or clearing, mPAM generates micrometer-resolution 3D images of paraffin- or agarose-embedded whole organs with high fidelity, achieved by label-free simultaneous sensing of DNA/RNA, hemoglobins, and lipids. mPAM provides histology-like imaging of cell nuclei, blood vessels, axons, and other anatomical structures, enabling the application of histopathological interpretation at the organelle level to analyze a whole organ. Its deep tissue imaging capability leads to less sectioning, resulting in negligible sectioning artifact. mPAM offers a new way to better understand complex biological organs.The state-of-the-art three-dimensional biomedical imaging often requires specific tissue preparation that may alter the physical properties of the specimen causing loss of information. Here Wong et al. develop a microtomy-assisted photoacoustic microscopy that allows imaging of biological samples without labelling agents and with reduced sectioning.

Use of a single xenon flash lamp for photoacoustic computed tomography of multiple-centimeter-thick biological tissue ex vivo and a whole mouse body in vivo

Abstract. While lasers have been commonly used as illumination sources in photoacoustic (PA) imaging, their high purchase and maintenance costs, as well as their bulkiness, have hindered the rapid clinical dissemination of PA imaging. With this in mind, we explore an alternative illumination source for PA tomography—a xenon flash lamp with high pulse energy and a microsecond pulse width. We demonstrate that, by using a single xenon flash lamp, we can image both a black latex cord placed in chicken breast tissue at a depth of up to 3.5 cm ex vivo and an entire mouse body in vivo. Our findings indicate that the xenon flash lamp, producing optical illumination that is safe for humans, can be potentially applied to human tissue imaging.

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.