The microcirculation plays a central role in the regulation of the metabolic, hemodynamic and thermal state of the individual [1
]. Many major diseases [2
] are manifest in the microcirculation before they are clinically evident, which provides a potential early perspective on the origin and progression of such diseases. However, established clinical imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasonography lack the resolution needed for microvascular imaging [9
]. Even with iodine contrast, x-ray imaging cannot image single capillaries. Thus, optical microscopy has been widely used to assess the cellular and molecular features of the microcirculation. Intravital microscopy (IVM), for example, is the gold standard for microcirculation studies. It 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 [10
], restricting its application to limited anatomical sites and interfering with the intrinsic microcirculatory function. Additionally, conventional IVM lacks depth resolution that is crucial for extracting the three-dimensional (3D) microvascular morphology. Confocal microscopy [11
] and two-photon microscopy [12
], noninvasive and possessing excellent depth-sectioning capability, have difficulty in detecting microvessels without exogenous fluorescent agents, which, although having greatly facilitated laboratory research, are still facing challenges in clinical translations [13
]. Orthogonal polarization spectral (OPS) imaging permits noninvasive microvascular imaging without the use of fluorescent dyes [14
], paving its way to the bedside. However, OPS provides no depth information and lacks the measurement consistency [15
] required for longitudinal studies.
To overcome these difficulties, we have developed optical-resolution photoacoustic microscopy (OR-PAM), a noninvasive volumetric microscopy technology capable of detecting the physiologically specific absorption signatures of endogenous chromophores, such as hemoglobin, in vivo
. The ability to introduce hemoglobin absorption contrast into the optical-resolution microscopy regime leads to an extremely versatile technique for microcirculation studies, without the limitations of fluorescent labeling and invasiveness. Besides the morphological parameters, such as vessel count, diameter, and length, OR-PAM also validates the quantification of important functional parameters, including total hemoglobin concentration (HbT) and hemoglobin oxygen saturation (sO2
] down to the capillary level. Multiple attractive features make OR-PAM a valuable tool for microcirculation studies (), which is demonstrated below by noninvasively monitoring microhemodynamic activities in vivo
Comparison of modern high-resolution microvascular imaging techniques. CM: confocal microscopy; TPM: two-photon microscopy.