Lidai Wang

Single-cell label-free photoacoustic flowoxigraphy in vivo

Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (CHb), oxygen saturation (sO2), sO2 gradient (), flow speed (vf), and oxygen release rate (rO2), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO2, , vf, and rO2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications.

Fast voice-coil scanning optical-resolution photoacoustic microscopy

We developed a photoacoustic imaging system that has real-time imaging capability with optical resolution. The imaging system is capable of scanning at 20 Hz over a 9 mm range and up to 40 Hz over a 1 mm scanning range. A focused laser beam provides a lateral resolution of 3.4 μm as measured in an optically nonscattering medium. Flows of micrometer-sized carbon particles or whole blood in a silicone tube and individual red blood cells (RBCs) in mouse ear capillaries were also imaged in real time, demonstrating the capability to image highly dynamic processes in vivo at a micrometer-scale resolution.

Multiscale photoacoustic tomography using reversibly switchable bacterial phytochrome as a near-infrared photochromic probe

Photoacoustic tomography (PAT) of genetically encoded probes allows for imaging of targeted biological processes deep in tissues with high spatial resolution; however, high background signals from blood can limit the achievable detection sensitivity. Here we describe a reversibly switchable nonfluorescent bacterial phytochrome for use in multiscale photoacoustic imaging, BphP1, with the most red-shifted absorption among genetically encoded probes. BphP1 binds a heme-derived biliverdin chromophore and is reversibly photoconvertible between red and near-infrared light-absorption states. We combined single-wavelength PAT with efficient BphP1 photoswitching, which enabled differential imaging with substantially decreased background signals, enhanced detection sensitivity, increased penetration depth and improved spatial resolution. We monitored tumor growth and metastasis with ∼100-μm resolution at depths approaching 10 mm using photoacoustic computed tomography, and we imaged individual cancer cells with a suboptical-diffraction resolution of ∼140 nm using photoacoustic microscopy. This technology is promising for biomedical studies at several scales.

Wide-field fast-scanning photoacoustic microscopy based on a water-immersible MEMS scanning mirror

By offering images with high spatial resolution and unique optical absorption contrast, optical-resolution photoacoustic microscopy (OR-PAM) has gained increasing attention in biomedical research. Recent developments in OR-PAM have improved its imaging speed, but have to sacrifice either the detection sensitivity or field of view or both. We have developed a wide-field fast-scanning OR-PAM by using a water-immersible microelectromechanical systems (MEMS) scanning mirror (MEMS-OR-PAM). In MEMS-OR-PAM, the optical and acoustic beams are confocally configured and simultaneously steered, which ensures the uniform detection sensitivity. A B-scan imaging speed as high as 400 Hz can be achieved over a 3 mm scanning range. Using the system, we imaged the flow dynamics of both red blood cells and carbon particles in a mouse ear in vivo. Presented results show that MEMS-OR-PAM could be a powerful tool for studying highly dynamic and time-sensitive biological phenomena.

Label-free photoacoustic nanoscopy

Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

Labeling Human Mesenchymal Stem Cells with Gold Nanocages for in vitro and in vivo Tracking by Two-Photon Microscopy and Photoacoustic Microscopy

Stem cell tracking is a highly important subject. Current techniques based on nanoparticle-labeling, such as magnetic resonance imaging, fluorescence microscopy, and micro-computed tomography, are plagued by limitations including relatively low sensitivity or penetration depth, involvement of ionizing irradiation, and potential cytotoxicity of the nanoparticles. Here we introduce a new class of contrast agents based on gold nanocages (AuNCs) with hollow interiors and porous walls to label human mesenchymal stem cells (hMSCs) for both in vitro and in vivo tracking using two-photon microscopy and photoacoustic microscopy. As demonstrated by the viability assay, the AuNCs showed negligible cytotoxicity under a reasonable dose, and did not alter the differentiation potential of the hMSCs into desired lineages. We were able to image the cells labeled with AuNCs in vitro for at least 28 days in culture, as well as to track the cells that homed to the tumor region in nude mice in vivo.

Single-cell photoacoustic thermometry

Abstract. A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community.

Single-impulse panoramic photoacoustic computed tomography of small-animal whole-body dynamics at high spatiotemporal resolution

Imaging of small animals has played an indispensable role in preclinical research by providing high dimensional physiological, pathological, and phenotypic insights with clinical relevance. Yet pure optical imaging suffers from either shallow penetration (up to ~1–2 mm) or a poor depth-to-resolution ratio (~1/3), and non-optical techniques for whole-body imaging of small animals lack either spatiotemporal resolution or functional contrast. Here, we demonstrate that standalone single-impulse photoacoustic computed tomography (SIP-PACT) mitigates these limitations by combining high spatiotemporal resolution (125-µm in-plane resolution, 50 µs / frame data acquisition and 50-Hz frame rate), deep penetration (48-mm cross-sectional width in vivo), anatomical, dynamical and functional contrasts, and full-view fidelity. By using SIP-PACT, we imaged in vivo whole-body dynamics of small animals in real time and obtained clear sub-organ anatomical and functional details. We tracked unlabeled circulating melanoma cells and imaged the vasculature and functional connectivity of whole rat brains. SIP-PACT holds great potential for both pre-clinical imaging and clinical translation.

Calibration-free quantification of absolute oxygen saturation based on the dynamics of photoacoustic signals

Photoacoustic tomography (PAT) is a hybrid imaging technique that has broad preclinical and clinical applications. Based on the photoacoustic effect, PAT directly measures specific optical absorption, which is the product of the tissue-intrinsic optical absorption coefficient and the local optical fluence. Therefore, quantitative PAT, such as absolute oxygen saturation (sO₂) quantification, requires knowledge of the local optical fluence, which can only be estimated through invasive measurements or sophisticated modeling of light transportation. In this Letter, we circumvent this requirement by taking advantage of the dynamics in sO₂. The new method works when the sO₂ transition can be simultaneously monitored with multiple wavelengths. For each wavelength, the ratio of photoacoustic amplitudes measured at different sO₂ states is utilized. Using the ratio cancels the contribution from optical fluence and allows calibration-free quantification of absolute sO₂. The new method was validated through both phantom and in vivo experiments.

Random-access optical-resolution photoacoustic microscopy using a digital micromirror device

We developed random-access optical-resolution photoacoustic microscopy using a digital micromirror device. This system can rapidly scan arbitrarily shaped regions of interest within a 40 μm×40 μm imaging area with a lateral resolution of 3.6 μm. To identify a region of interest, a global structural image is first acquired, then the selected region is scanned. The random-access ability was demonstrated by imaging two static samples, a carbon fiber cross and a monolayer of red blood cells, with an acquisition rate up to 4 kHz. The system was then used to monitor blood flow in vivo in real time within user-selected capillaries in a mouse ear. By imaging only the capillary of interest, the frame rate was increased by up to 9.2 times.