Chenghung Yeh

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.

Fully motorized optical-resolution photoacoustic microscopy

We have developed fully motorized optical-resolution photoacoustic microscopy (OR-PAM), which integrates five complementary scanning modes and simultaneously provides a high imaging speed and a wide field of view (FOV) with 2.6 μm lateral resolution. With one-dimensional (1D) motion-mode mechanical scanning, we measured the blood flow through a cross section of a blood vessel in vivo. With two-dimensional (2D) optical scanning at a laser repetition rate of 40 kHz, we achieved a 2 kHz B-scan rate over a range of 50 μm with 20 A-lines and 50 Hz volumetric-scan rate over a FOV of 50  μm × 50  μm with 400 A-lines, which enabled real-time tracking of cellular dynamics in vivo. With synchronized 1D optical and 2D mechanical hybrid scanning, we imaged a 10  mm × 8  mm FOV within three minutes, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical contour scanning, we maintained the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is essential for quantitative studies.

Photoacoustic microscopy of blood pulse wave

Blood pulse wave velocity (PWV) is an important physiological parameter that characterizes vascular stiffness. In this letter, we present electrocardiogram-synchronized, photoacoustic microscopy for noninvasive quantification of the PWV in the peripheral vessels of living mice. Interestingly, blood pulse wave-induced fluctuations in blood flow speed were clearly observed in arteries and arterioles, but not in veins or venules. Simultaneously recorded electrocardiograms served as references to measure the travel time of the pulse wave between two cross sections of a chosen vessel and vessel segmentation analysis enabled accurate quantification of the travel distance. PWVs were quantified in ten vessel segments from two mice. Statistical analysis shows a linear correlation between the PWV and the vessel diameter which agrees with known physiology.

In vivo optically encoded photoacoustic flowgraphy.

We present an optically encoded photoacoustic (PA) flow imaging method based on optical-resolution PA microscopy. An intensity-modulated continuous-wave laser photothermally encodes the flowing medium, and a pulsed laser generates PA waves to image the encoded heat pattern. Flow speeds can be calculated by cross correlation. The method was validated in phantoms at flow speeds ranging from 0.23 to 11  mm/s. Venous blood flow speed in a mouse ear was also measured.

Three-dimensional arbitrary trajectory scanning photoacoustic microscopy.

We have enhanced photoacoustic microscopy with three-dimensional arbitrary trajectory (3-DAT) scanning, which can rapidly image selected vessels over a large field of view (FOV) and maintain a high signal-to-noise ratio (SNR) despite the depth variation of the vessels. We showed that hemoglobin oxygen saturation (sO2 ) and blood flow can be measured simultaneously in a mouse ear in vivo at a frame rate 67 times greater than that of a traditional two-dimensional raster scan. We also observed sO2 dynamics in response to switching from systemic hypoxia to hyperoxia. 3-DAT-scanning photoacoustic microscopy. Schematic diagram of the 3D scanning stage and method.

Microvascular quantification based on contour-scanning photoacoustic microscopy

Abstract. Accurate quantification of microvasculature remains of interest in fundamental pathophysiological studies and clinical trials. Current photoacoustic microscopy can noninvasively quantify properties of the microvasculature, including vessel density and diameter, with a high spatial resolution. However, the depth range of focus (i.e., focal zone) of optical-resolution photoacoustic microscopy (OR-PAM) is often insufficient to encompass the depth variations of features of interest—such as blood vessels—due to uneven tissue surfaces. Thus, time-consuming image acquisitions at multiple different focal planes are required to maintain the region of interest in the focal zone. We have developed continuous three-dimensional motorized contour-scanning OR-PAM, which enables real-time adjustment of the focal plane to track the vessels’ profile. We have experimentally demonstrated that contour scanning improves the signal-to-noise ratio of conventional OR-PAM by as much as 41% and shortens the image acquisition time by 3.2 times. Moreover, contour-scanning OR-PAM more accurately quantifies vessel density and diameter, and has been applied to studying tumors with uneven surfaces.

Optical-resolution photoacoustic microscopy of the metabolic rate of oxygen in a mouse renal tumor model

We propose using noninvasive longitudinal optical-resolution photoacoustic microscopy (L-ORPAM) to quantify blood flow flux, oxygen saturation (sO2), and thereby the metabolic rate of oxygen (MRO2), for a renal tumor model in the same mouse over weeks to months. Experiments showed that the sO2 difference between the artery and vein decreased greatly due to the arteriovenous shunting effect during tumor growth. Moreover, hypermetabolism was exhibited by an increase in MRO2.

Direct measurement of hypoxia in a xenograft multiple myeloma model by optical-resolution photoacoustic microscopy

ABSTRACT Using photoacoustic microscopy (PAM), we evaluated non-invasively oxygenation and vascularization in vivo due to multiple myeloma (MM) progression. Mice injected with MM.1S-GFP were monitored with a fluorescence microscope for tumor progression. In vivo PAM of the cerebral bone marrow quantified the total oxygen saturation (sO2). At 28 days after the MM cell injection, the total sO2 had decreased by half in the developing tumor regions, while in the non-tumor regions it had decreased by 20% compared with the value at one day post MM injection. The blood vessel density was reduced by 35% in the developing tumor regions, while in the non-tumor regions it was reduced by 8% compared with the value at one day post MM injection. Hence, PAM corroborated the development of hypoxia due to MM progression and demonstrated decreased vascularization surrounding the tumor areas.

Dry coupling for whole-body small-animal photoacoustic computed tomography

Abstract. We have enhanced photoacoustic computed tomography with dry acoustic coupling that eliminates water immersion anxiety and wrinkling of the animal and facilitates incorporating complementary modalities and procedures. The dry acoustic coupler is made of a tubular elastic membrane enclosed by a closed transparent water tank. The tubular membrane ensures water-free contact with the animal, and the closed water tank allows pressurization for animal stabilization. The dry coupler was tested using a whole-body small-animal ring-shaped photoacoustic computed tomography system. Dry coupling was found to provide image quality comparable to that of conventional water coupling.

Label-free hypoxia measurement in a xenograft multiple myeloma model using optical-resolution photoacoustic microscopy (Conference Presentation)

In cancer research, regions of increasingly lowered oxygenation in tissue (hypoxia), which are due to tumor development, are considered to play an important role in activating various signaling pathways that facilitate further development of the cancer. However, devising a minimally invasive method to monitor tissue oxygenation has remained a challenge. Photoacoustic microscopy has been posed as a solution in a variety of preclinical research studies. Here, using optical-resolution photoacoustic microscopy (OR-PAM), for the first time, we non-invasively measured oxygenation and vascularization in vivo caused by multiple myeloma (MM) progression. Mice injected with MM cells tagged with green fluorescent protein were monitored with a fluorescence microscope for tumor progression over the course of 28 days. OR-PAM evaluated the oxygen saturation (sO2) and the blood vessel density in the cerebral bone marrow, where MM cells home. At 28 days after the injection of MM cells, the total sO2 had dropped by 50% in the developing tumor regions, while in the non-tumor developing regions it had dropped by 20% compared with the value at one day after MM injection. The blood vessel density had dropped by 35% in the tumor developing regions, while in the non-tumor developing regions it had dropped by 8% compared with the value at one day after MM injection. In summary, non-invasive measurement by OR-PAM correlated the development of hypoxia with to MM progression. It revealed decreased vascularization surrounding the tumor areas, which we feel can be ascribed to the rapid tumor progression.