Photoacoustic imaging refers to imaging based on the photoacoustic effect. The object is irradiated by a short-pulsed laser beam. An intensity-modulated laser beam is an alternative. Light absorbed by the object is partially converted into heat, in most cases with a fraction of approximately (1 – fluorescence quantum yield). The heat is further converted to a pressure rise via thermo-elastic expansion. The pressure rise propagates as an ultrasonic wave, referred to as a photoacoustic wave. The photoacoustic wave is detected by ultrasonic transducers. The detected signals are used by a computer to form an image.
The main function of the cardiovascular system is to provide, through perfused vascular beds, the necessities for living tissues and organs. The distributing arterial trees transport oxygen, humoral agents, and nutrients to the vital parts of the body; in the mean time, the venous trees collect metabolic wastes. Microcirculation, the distal functional unit of the cardiovascular system, provides exchange sites for gases, nutrients, metabolic wastes, and thermal energy between the blood and the tissues. Pathologic microcirculation reflects the breakdown of homeostasis in organisms, which ultimately leads to tissue inviability. Thus, in vivo microvascular imaging and characterization is of significant physiological, pathophysiological, and clinical importance.
Well‐established clinical imaging modalities such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and ultrasound imaging have been adopted for vascular imaging. However, these techniques often lack either sufficient spatial resolution or satisfactory contrast (or both) to be effective for microvascular imaging. To bridge this gap, optical microscopy has been widely used to dissect the delicate features of the microvasculature. Intravital microscopy (IVM), the gold standard for microcirculation studies, allows quantification of vessel count, diameter, length, density, permeability, and blood flow velocity. Nevertheless, to observe capillaries in vivo, IVM generally requires trans-illumination and surgical preparation, which disturb the intrinsic microvasculature and are restricted to limited anatomical sites. Moreover, conventional IVM lacks the depth resolution that is crucial for characterizing three-dimensional (3D) microvascular morphology. Confocal microscopy and two-photon microscopy (TPM) avoid such invasive preparation and enable volumetric visualization of the microvasculature. However, their imaging contrasts come primarily from exogenous fluorescent agents (hemoglobin is nearly non-fluorescent), which—though having been very successful in laboratory research—are still facing challenges in clinical translations. Orthogonal polarization spectral (OPS) imaging enables intrinsic microvascular imaging; however, it provides only a two-dimensional (2D) visualization of the microvasculature and lacks the measurement consistency required for chronic studies.Doppler optical coherence tomography (D-OCT) demonstrates the intrinsic imaging of microvascular perfusion by extracting blood flow information, but its resolution and sensitivity are not yet sufficient to image single capillaries. More importantly, among the mainstream techniques mentioned above, only OPS can assess the functional parameter of total hemoglobin concentration (HbT), and none of them have direct access to hemoglobin oxygen saturation (sO2)—another important functional parameter. To measure sO2 in vivo: (i) Raman spectroscopy has been integrated into IVM; however, the resonance Raman signals generated in tissue compartments other than blood could interfere with the analysis of hemoglobin. (ii) Hyperspectral imaging has been integrated into optical coherence tomography (OCT); nevertheless, it requires transillumination and only provides 2D mapping. (iii) Recently, TPM has made exciting progress in terms of label-free imaging.With TPM, microvascular morphology and oxygenation have been imaged in vivo without fluorescence labeling; however, sO2 quantification is not available yet due to the limited signal-to-noise ratio (SNR). Therefore, a novel technology is needed to overcome these limitations.
Photoacoustic microscopy is the fastest growing biomedical imaging technology, which affords high-resolution sensing of rich optical contrast in vivo at super-depths—depths beyond the optical transport mean free path (~1 mm in the skin). Although commercially available high-resolution three-dimensional optical imaging modalities—including confocal microscopy, two-photon microscopy and optical coherence tomography—have fundamentally impacted biomedicine,none can reach super-depths in tissue. In PAM, short-pulsed laser light is absorbed by biological tissue and converted to transient heating, which is subsequently converted into an ultrasonic wave due to thermoelastic expansion. Detection of the ultrasonic wave yields a tomographic image. Taking advantage of low ultrasonic scattering, PAM equivalently improves tissue optical transparency by a factor of 1000 and consequently enables super-depth penetration at high resolution. As a unique feature, PAM is exquisitely sensitive to optical absorption, most sensitive among all imaging modalities. PAM enables in vivo imaging of the smallest blood vessels (i.e., capillaries) and single cells with intrinsic tissue contrast (i.e., no exogenous contrast agent injected into the tissue). Both functional and molecular imaging based on optical contrast have been achieved by PAM.