Bin Rao

Label-free photoacoustic ophthalmic angiography

We present label-free functional photoacoustic imaging of the ocular microvasculature in living animals. The anterior segment of an adult mouse was imaged with a laser exposure level well within the American National Standards Institute safety standards. Individual red blood cells traveling along the iris capillaries were clearly resolved, and the hemoglobin oxygen saturation in the iris microvasculature was imaged spectrally. We believe that photoacoustic imaging has the potential to advance the diagnosis and treatment of ocular diseases in humans.

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.

Hybrid-scanning optical-resolution photoacoustic microscopy for in vivo vasculature imaging

Recently developed optical-resolution photoacoustic microscopy (OR-PAM), which is based on the detection of optical absorption contrast, is complementary to other optical microscopy modalities such as optical confocal microscopy, optical coherence tomography, and multiphoton microscopy. A hybrid optical-mechanical scanning configuration increases the imaging speed of OR-PAM significantly, facilitating many demanding biomedical applications. With a high-pulse-repetition-rate laser, the hybrid-scanning OR-PAM can acquire one-dimensional depth-resolved images (A-lines) at 5 kHz and two-dimensional B-scan images containing 800 A-lines at 6.25 Hz. We demonstrated in vivo in a mouse three-dimensional imaging of the iris vasculature in 128 s for an 800x800x200 data set and of the ear vasculature in 256 s for an 800x1600x200 data set.

Real-time four-dimensional optical-resolution photoacoustic microscopy with Au nanoparticle-assisted subdiffraction-limit resolution

Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed, high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per second with repeated two-dimensional (2D) raster scans of 100 by 50 points. The corresponding 2D B-scan (50 A-lines) frame rate was 1800 Hz, and the one-dimensional A-scan rate was 90,000 Hz. The lateral resolution is 0.23 ± 0.03 μm for Au nanowire imaging, which is 2.0 times below the diffraction limit.

Optical-resolution photoacoustic endomicroscopy in vivo

Optical-resolution photoacoustic microscopy (OR-PAM) has become a major experimental tool of photoacoustic tomography, with unique imaging capabilities for various biological applications. However, conventional imaging systems are all table-top embodiments, which preclude their use in internal organs. In this study, by applying the OR-PAM concept to our recently developed endoscopic technique, called photoacoustic endoscopy (PAE), we created an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system, which enables internal organ imaging with a much finer resolution than conventional acoustic-resolution PAE systems. OR-PAEM has potential preclinical and clinical applications using either endogenous or exogenous contrast agents.

Photoacoustic and optical coherence tomography of epilepsy with high temporal and spatial resolution and dual optical contrasts

Epilepsy mapping with high spatial and temporal resolution has great significance for both fundamental research on epileptic neurons and the clinical management of epilepsy. In this communication, we demonstrate for the first time in vivo epilepsy mapping with high spatial and temporal resolution and dual optical contrasts in an animal model. Through the variations of a depthresolved optical coherence tomography signal with optical scattering contrast, we observed that epileptic neuron activities modulated the optical refractive index of epileptic neurons and their surrounding tissue. Simultaneously, through neurovasculature coupling mechanisms and optical absorption contrast, we used photoacoustic signals to document the hemodynamic changes of the microvasculature surrounding the epileptic neurons. The epilepsy mapping results were confirmed by a simultaneously recorded electroencephalogram signal during epileptic seizure. Our new epilepsy mapping tool, with high temporal and spatial resolution and dual optical contrasts, may find many applications, such as drug development and epilepsy surgery.

Integrated photoacoustic, confocal, and two-photon microscope

Abstract. The invention of green fluorescent protein and other molecular fluorescent probes has promoted applications of confocal and two-photon fluorescence microscopy in biology and medicine. However, exogenous fluorescence contrast agents may affect cellular structure and function, and fluorescence microscopy cannot image nonfluorescent chromophores. We overcome this limitation by integrating optical-resolution photoacoustic microscopy into a modern Olympus IX81 confocal, two-photon, fluorescence microscope setup to provide complementary, label-free, optical absorption contrast. Automatically coregistered images can be generated from the same sample. Imaging applications in ophthalmology, developmental biology, and plant science are demonstrated. For the first time, in a familiar microscopic fluorescence imaging setting, this trimodality microscope provides a platform for future biological and medical discoveries.

Fast-scanning reflection-mode integrated photoacoustic and optical-coherence microscopy

We previously demonstrated that multimodal microscopy combining photoacoustic microscopy and optical coherence tomography can provide comprehensive insight into biological tissue at μm-level resolution by exploiting both optical absorption and scattering contrasts. Recently, we have developed a second-generation integrated photoacoustic and optical-coherence microscope, which can potentially be adapted for clinical applications. In this new system, we can perform photoacoustic and optical-coherence imaging simultaneously at a speed of 5,000 A-lines per second with real-time on-screen display. Also, both modalities now work in reflection mode instead of transmission mode, allowing easy access to various anatomical locations of interest. Imaging of skin and eye has been demonstrated in living small animals.

Photoacoustic imaging of voltage responses beyond the optical diffusion limit

Non-invasive optical imaging of neuronal voltage response signals in live brains is constrained in depth by the optical diffusion limit, which is due primarily to optical scattering by brain tissues. Although photoacoustic tomography breaks this limit by exciting the targets with diffused photons and detecting the resulting acoustic responses, it has not been demonstrated as a modality for imaging voltage responses. In this communication, we report the first demonstration of photoacoustic voltage response imaging in both in vitro HEK-293 cell cultures and in vivo mouse brain surfaces. Using spectroscopic photoacoustic tomography at isosbestic wavelengths, we can separate voltage response signals and hemodynamic signals on live brain surfaces. By imaging HEK-293 cell clusters through 4.5 mm thick ex vivo rat brain tissue, we demonstrate photoacoustic tomography of cell membrane voltage responses beyond the optical diffusion limit. Although the current voltage dye does not immediately allow in vivo deep brain voltage response imaging, we believe our method opens up a feasible technical path for deep brain studies in the future.

Photoacoustic microscopy of human teeth

Photoacoustic microscopy (PAM) utilizes short laser pulses to deposit energy into light absorbers and sensitively detects the ultrasonic waves the absorbers generate in response. PAM directly renders a three-dimensional spatial distribution of sub-surface optical absorbers. Unlike other optical imaging technologies, PAM features label-free optical absorption contrast and excellent imaging depths. Standard dental imaging instruments are limited to X-ray and CCD cameras. Subsurface optical dental imaging is difficult due to the highly-scattering enamel and dentin tissue. Thus, very few imaging methods can detect dental decay or diagnose dental pulp, which is the innermost part of the tooth, containing the nerves, blood vessels, and other cells. Here, we conducted a feasibility study on imaging dental decay and dental pulp with PAM. Our results showed that PAM is sensitive to the color change associated with dental decay. Although the relative PA signal distribution may be affected by surface contours and subsurface reflections from deeper dental tissue, monitoring changes in the PA signals (at the same site) over time is necessary to identify the progress of dental decay. Our results also showed that deep-imaging, near-infrared (NIR) PAM can sensitively image blood in the dental pulp of an in vitro tooth. In conclusion, PAM is a promising tool for imaging both dental decay and dental pulp.