Konstantin Maslov

Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging.

Although optical absorption is strongly associated with the physiological status of biological tissue, existing high-resolution optical imaging modalities, including confocal microscopy, two-photon microscopy and optical coherence tomography, do not sense optical absorption directly. Furthermore, optical scattering prevents these methods from imaging deeper than ∼1 mm below the tissue surface. Here we report functional photoacoustic microscopy (fPAM), which provides multiwavelength imaging of optical absorption and permits high spatial resolution beyond this depth limit with a ratio of maximum imaging depth to depth resolution greater than 100. Reflection mode, rather than orthogonal or transmission mode, is adopted because it is applicable to more anatomical sites than the others. fPAM is demonstrated with in vivo imaging of angiogenesis, melanoma, hemoglobin oxygen saturation (sO2) of single vessels in animals and total hemoglobin concentration in humans.

Optical-resolution photoacoustic microscopy for in vivo imaging of single capillaries

Capillaries, the smallest blood vessels, are the distal end of the vasculature where oxygen and nutrients are exchanged between blood and tissue. Hence, noninvasive imaging of capillaries and function in vivo has long been desired as a window to studying fundamental physiology, such as neurovascular coupling. Existing imaging modalities cannot provide the required sensitivity and spatial resolution. We present in vivo imaging of the microvasculature including single capillaries in mice using optical-resolution photoacoustic microscopy (OR-PAM) developed in our laboratory. OR-PAM provides a lateral resolution of 5 microm and an imaging depth >0.7 mm.

In vivo dark-field reflection-mode photoacoustic microscopy

Reflection-mode photoacoustic microscopy with dark-field laser pulse illumination and high-numerical-aperture ultrasonic detection is designed and implemented in noninvasively imaged blood vessels in the skin in vivo. Dark-field optical illumination minimizes the interference caused by strong photoacoustic signals from superficial structures. A high-numerical-aperture acoustic lens provides high lateral resolution, 45-120 microm in this system. A broadband ultrasonic detection system provides high axial resolution, estimated to be approximately 15 microm. The optical illumination and ultrasonic detection are in a coaxial confocal configuration for optimal image quality. The system is capable of imaging optical-absorption contrast as deep as 3 mm in biological tissue.

Imaging of hemoglobin oxygen saturation variations in single vessels in vivo using photoacoustic microscopy

Photoacoustic microscopy was used to noninvasively image variations in hemoglobin oxygen saturation (SO_2) in the subcutaneous microvasculature of rats in vivo. In phantom tests, the calculated concentration fractions of red ink in double-ink mixtures matched the actual values with a 1% error. In ex vivo studies, the calculated SO_2 in bovine blood agreed with the standard spectrophotometric measurements within a 4% systematic difference. In in vivo studies, arteries and veins were separated based on the measured SO_2 values and variations in SO_2 between different physiological states (hyperoxia, normoxia, and hypoxia) were imaged in single blood vessels.

Second-generation optical-resolution photoacoustic microscopy with improved sensitivity and speed

We developed second-generation (G2) optical-resolution photoacoustic microscopy (OR-PAM). Incorporation of a novel acoustic detection scheme improved upon the sensitivity of our first-generation (G1) system by 18.4 dB, deepening the in vivo tissue penetration to 1.2 mm at 570 nm. Moreover, translating the imaging head instead of the living object accelerated the scanning speed by a factor of 5, widening the field of view within the same acquisition time. Mouse ears, as well as mouse brains with intact craniums, were imaged in vivo in both total concentration and oxygen saturation of hemoglobin.

Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo

At present, clinicians routinely apply ultrasound endoscopy in a variety of interventional procedures that provide treatment solutions for diseased organs. Ultrasound endoscopy not only produces high-resolution images, but also is safe for clinical use and broadly applicable. However, for soft tissue imaging, its mechanical wave–based image contrast fundamentally limits its ability to provide physiologically specific functional information. By contrast, photoacoustic endoscopy possesses a unique combination of functional optical contrast and high spatial resolution at clinically relevant depths, ideal for imaging soft tissues. With these attributes, photoacoustic endoscopy can overcome the current limitations of ultrasound endoscopy. Moreover, the benefits of photoacoustic imaging do not come at the expense of existing ultrasound functions; photoacoustic endoscopy systems are inherently compatible with ultrasound imaging, thereby enabling multimodality imaging with complementary contrast. Here we present simultaneous photoacoustic and ultrasonic dual-mode endoscopy and show its ability to image internal organs in vivo, thus illustrating its potential clinical application.

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.

Three-dimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy

Dual-wavelength reflection-mode photoacoustic microscopy is used to noninvasively obtain three-dimensional (3-D) images of subcutaneous melanomas and their surrounding vasculature in nude mice in vivo. The absorption coefficients of blood and melanin-pigmented melanomas vary greatly relative to each other at these two optical wavelengths (764 and 584 nm). Using high-resolution and high-contrast photoacoustic imaging in vivo with a near-infrared (764-nm) light source, the 3-D melanin distribution inside the skin is imaged, and the maximum thickness of the melanoma (approximately 0.5 mm) is measured. The vascular system surrounding the melanoma is also imaged with visible light (584 nm) and the tumor-feeding vessels found. This technique can potentially be used for melanoma diagnosis, prognosis, and treatment planning.

Label-free oxygen-metabolic photoacoustic microscopy in vivo

Almost all diseases, especially cancer and diabetes, manifest abnormal oxygen metabolism. Accurately measuring the metabolic rate of oxygen (MRO(2)) can be helpful for fundamental pathophysiological studies, and even early diagnosis and treatment of disease. Current techniques either lack high resolution or rely on exogenous contrast. Here, we propose label-free metabolic photoacoustic microscopy (mPAM) with small vessel resolution to noninvasively quantify MRO(2) in vivo in absolute units. mPAM is the unique modality for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed. Hyperthermia, cryotherapy, melanoma, and glioblastoma were longitudinally imaged in vivo. Counterintuitively, increased MRO(2) does not necessarily cause hypoxia or increase oxygen extraction. In fact, early-stage cancer was found to be hyperoxic despite hypermetabolism.

Sentinel Lymph Nodes in the Rat: Noninvasive Photoacoustic and US Imaging with a Clinical US System

PURPOSE To evaluate in vivo sentinel lymph node (SLN) mapping by using photoacoustic and ultrasonographic (US) imaging with a modified clinical US imaging system. MATERIALS AND METHODS Animal protocols were approved by the Animal Studies Committee. Methylene blue dye accumulation in axillary lymph nodes of seven healthy Sprague-Dawley rats was imaged by using a photoacoustic imaging system adapted from a clinical US imaging system. To investigate clinical translation, the imaging depth was extended up to 2.5 cm by adding chicken or turkey breast on top of the rat skin surface. Three-dimensional photoacoustic images were acquired by mechanically scanning the US transducer and light delivery fiber bundle along the elevational direction. RESULTS Photoacoustic images of rat SLNs clearly help visualization of methylene blue accumulation, whereas coregistered photoacoustic/US images depict lymph node positions relative to surrounding anatomy. Twenty minutes following methylene blue injection, photoacoustic signals from SLN regions increased nearly 33-fold from baseline signals in preinjection images, and mean contrast between SLNs and background tissue was 76.0 +/- 23.7 (standard deviation). Methylene blue accumulation in SLNs was confirmed photoacoustically by using the optical absorption spectrum of the dye. Three-dimensional photoacoustic images demonstrate dynamic accumulation of methylene blue in SLNs after traveling through lymph vessels. CONCLUSION In vivo photoacoustic and US mapping of SLNs was successfully demonstrated with a modified clinical US scanner. These results raise confidence that photoacoustic and US imaging can be used clinically for accurate, noninvasive imaging of SLNs for axillary lymph node staging in breast cancer patients.