Functional Photoacoustic Microscopy Research Articles

Imaging of temperature dependent hemodynamics in the rat sciatic nerve by functional photoacoustic microscopy

BackgroundVascular hemodynamics is central to the regulation of neuro-metabolism and plays important roles in peripheral nerves diseases and their prevention. However, at present there are only a few techniques capable of directly measuring peripheral nerve vascular hemodynamics.MethodHere, we investigate the use of dark-field functional photoacoustic microscopy (fPAM) for intrinsic visualizing of the relative hemodynamics of the rat sciatic nerve in response to localized temperature modulation (i.e., cooling and rewarming).Results and conclusionOur main results show that the relative functional total hemoglobin concentration (HbT) is more significantly correlated with localized temperature changes than the hemoglobin oxygen saturation (SO2) changes in the sciatic nerve. Our study also indicates that the relative HbT changes are better markers of neuronal activation than SO2 during nerve temperature changes. Our results show that fPAM is a promising candidate for in vivo imaging of peripheral nerve hemodynamics without the use of contrast agents. Additionally, this technique may shed light on the neuroprotective effect of hypothermia on peripheral nerves by visualizing their intrinsic hemodynamics.

Neurovascular coupling: in vivo optical techniques for functional brain imaging.

Optical imaging techniques reflect different biochemical processes in the brain, which is closely related with neural activity. Scientists and clinicians employ a variety of optical imaging technologies to visualize and study the relationship between neurons, glial cells and blood vessels. In this paper, we present an overview of the current optical approaches used for the in vivo imaging of neurovascular coupling events in small animal models. These techniques include 2-photon microscopy, laser speckle contrast imaging (LSCI), voltage-sensitive dye imaging (VSDi), functional photoacoustic microscopy (fPAM), functional near-infrared spectroscopy imaging (fNIRS) and multimodal imaging techniques. The basic principles of each technique are described in detail, followed by examples of current applications from cutting-edge studies of cerebral neurovascular coupling functions and metabolic. Moreover, we provide a glimpse of the possible ways in which these techniques might be translated to human studies for clinical investigations of pathophysiology and disease. In vivo optical imaging techniques continue to expand and evolve, allowing us to discover fundamental basis of neurovascular coupling roles in cerebral physiology and pathophysiology.

Video-rate functional photoacoustic microscopy at depths

We report the development of functional photoacoustic microscopy capable of video-rate high-resolution in vivo imaging in deep tissue. A lightweight photoacoustic probe is made of a single-element broadband ultrasound transducer, a compact photoacoustic beam combiner, and a bright-field light delivery system. Focused broadband ultrasound detection provides a 44-μm lateral resolution and a 28-μm axial resolution based on the envelope (a 15-μm axial resolution based on the raw RF signal). Due to the efficient bright-field light delivery, the system can image as deep as 4.8 mm in vivo using low excitation pulse energy (28 μJ per pulse, 0.35 mJ/cm² on the skin surface). The photoacoustic probe is mounted on a fast-scanning voice-coil scanner to acquire 40 two-dimensional (2-D) B-scan images per second over a 9-mm range. High-resolution anatomical imaging is demonstrated in the mouse ear and brain. Via fast dual-wavelength switching, oxygen dynamics of mouse cardio-vasculature is imaged in realtime as well.

Investigation of the cerebral hemodynamic response function in single blood vessels by functional photoacoustic microscopy

The specificity of the hemodynamic response function (HRF) is determined spatially by the vascular architecture and temporally by the evolution of hemodynamic changes. Here, we used functional photoacoustic microscopy (fPAM) to investigate single cerebral blood vessels of rats after left forepaw stimulation. In this system, we analyzed the spatiotemporal evolution of the HRFs of the total hemoglobin concentration (HbT), cerebral blood volume (CBV), and hemoglobin oxygen saturation (SO(2)). Changes in specific cerebral vessels corresponding to various electrical stimulation intensities and durations were bilaterally imaged with 36 × 65-μm(2) spatial resolution. Stimulation intensities of 1, 2, 6, and 10 mA were applied for periods of 5 or 15 s. Our results show that the relative functional changes in HbT, CBV, and SO(2) are highly dependent not only on the intensity of the stimulation, but also on its duration. Additionally, the duration of the stimulation has a strong influence on the spatiotemporal characteristics of the HRF as shorter stimuli elicit responses only in the local vasculature (smaller arterioles), whereas longer stimuli lead to greater vascular supply and drainage. This study suggests that the current fPAM system is reliable for studying relative cerebral hemodynamic changes, as well as for offering new insights into the dynamics of functional cerebral hemodynamic changes in small animals.

Highlights: Transcranial Imaging of Functional Cerebral Hemodynamic Changes in Single Blood Vessels

Speeding up the functional brain imaging is the holy grail, or appropriately the ‘holy technology', of cognitive neurosciences because of its compelling potential to localize and investigate functional or cognitive brain responses to realistic, rapidly presented stimuli. Advances in functional magnetic resonance imaging continue to result in improved spatial resolution, but temporal resolution—imaging the early response to any stimulus—remains a challenge. This is where a novel optical technique that improves temporal resolution would come in handy. Optical methods may offer added benefits of lower costs and potential to carrying out continuous, long-term functional imaging. The manuscript by Liao et al (2012) describes a novel functional photoacoustic microscopy technique for detecting multiparameter functional responses evoked by peripheral stimuli. Functional photoacoustic microscopy is able to measure the total hemoglobin concentration (HbT), cerebral blood volume, hemoglobin oxygen saturation (SO2), and the transient hemodynamic response in single cortical vessels of mice with intact skulls. This transcranial imaging technique complements other existing neuroimaging approaches for longitudinal investigation of the hemodynamic response with high temporal resolution. It uses the optimal balance between spatial resolution, temporal resolution, and depth of imaging that is possible with ultrasonic imaging to achieve high intrinsic optical contrast while imaging through an intact skull. Thus, the method shows tremendous potential for small animal imaging for basic science research. Optical intrinsic signal imaging studies have consistently encountered a brief stimulus-evoked darkening, dubbed the initial dip, followed by a strong brightening when imaged under illumination that is preferentially absorbed by deoxyhemoglobin (HbR) (Grinvald et al, 1986; Malonek and Grinvald, 1996). In parallel, some functional magnetic resonance imaging studies have reported an initial dip in the blood oxygen level-dependent signal before its eventual rise (Menon et al, 1995; Kim et al, 2000). The initial dip in the blood oxygen level-dependent signal affords the promise of speeding up the imaging responses to rapidly delivered stimuli by shortening the image acquisition time. However, there are still a number of unclear issues some questions about the interpretation of the initial dip which has been robustly and reliably distinguished in optical intrinsic signal imaging, but proven to be elusive in functional magnetic resonance imaging (Ances, 2004; Buxton, 2001). This paper shows for the first time the consistent occurrence of the initial dip phenomenon using the new functional photoacoustic microscopy system. The results also display more localized blood flow changes in the cerebral arterioles, but no significant initial dip was observed in the superior sagittal sinus. Thus, further experimentation and validation are needed before functional photoacoustic microscopy becomes a universal method of imaging vascular responses to evoked neuronal activity. Overall, this innovative work serves two purposes. First, it establishes the capabilities and utilities of the noninvasive photoacoustic microscopy system for brain function studies. Second, it contributes to the understanding of vascular response to functional stimuli. It is important that this work be replicated by other investigators; this highlight is an encouragement to just do that. In addition, the investigators and the scientific community are encouraged to show compelling applications of this technology that evidently complements the other methodologies ranging from optical intrinsic signal imaging to blood oxygen level-dependent functional magnetic resonance imaging.

Transcranial Imaging of Functional Cerebral Hemodynamic Changes in Single Blood Vessels using in vivo Photoacoustic Microscopy

Optical imaging of changes in total hemoglobin concentration (HbT), cerebral blood volume (CBV), and hemoglobin oxygen saturation (SO(2)) provides a means to investigate brain hemodynamic regulation. However, high-resolution transcranial imaging remains challenging. In this study, we applied a novel functional photoacoustic microscopy technique to probe the responses of single cortical vessels to left forepaw electrical stimulation in mice with intact skulls. Functional changes in HbT, CBV, and SO(2) in the superior sagittal sinus and different-sized arterioles from the anterior cerebral artery system were bilaterally imaged with unambiguous 36 × 65-μm(2) spatial resolution. In addition, an early decrease of SO(2) in single blood vessels during activation (i.e., 'the initial dip') was observed. Our results indicate that the initial dip occurred specifically in small arterioles of activated regions but not in large veins. This technique complements other existing imaging approaches for the investigation of the hemodynamic responses in single cerebral blood vessels.

Early monitoring of cerebral hypoperfusion in rats by laser speckle imaging and functional photoacoustic microscopy

Because cerebral hypoperfusion brings damage to the brain, prevention of cerebrovascular diseases correlative to hypoperfusion by studying animal models makes great sense. Since complicated cerebrovascular adaptive changes in hypoperfusion could not be revealed only by cerebral blood flow (CBF) velocity imaging, we performed multi-parameter imaging by combining laser speckle imaging and functional photoacoustic microscopy. The changes in CBF, hemoglobin oxygen saturation (SO(2)), and total hemoglobin concentration (HbT) in single blood vessels of ipsilateral cortex were observed during transient cerebral hypoperfusion by ligating the unilateral common carotid artery in rats. CBF, SO(2), and HbT, respectively, decreased to 37 ± 3%, 71 ± 7.5%, and 92 ± 1.3% of baseline in 6 s immediately after occlusion, and then recovered to 77 ± 4.8%, 84 ± 8%, and 96 ± 2% of baseline in 60 s. These parameters presented the decrease with different degree and the following recovery over time after ligation, the recovery of SO(2) lagged behind those of CBF and HbT, which had the similar response. The results demonstrated that complete monitoring of both cerebral hemodynamic response and oxygen metabolic changes occurred at the earliest period of cerebral hypoperfusion was possible by using the two image modalities with high temporal and spatial resolution.

Functional photoacoustic microscopy of diabetic vasculature

We used functional photoacoustic microscopy to image diabetes-induced damage to the microvasculature. To produce an animal model for Type 1 diabetes, we used streptozotocin (STZ), which is particularly toxic to the insulin-producing beta cells of the pancreas in mammals. A set number of ND4 Swiss Webster mice received intraperitoneal injections of STZ for five consecutive days at 50 mg/kg. Most mice developed a significant rise in blood glucose level (≈ 400 mg/dL) within three weeks of the first injection. Changes in vasculature and hemodynamics were monitored for six weeks. The mouse ear was imaged with an optical-resolution photoacoustic microscope at a main blood vessel branch from the root of the ear. There are noticeable and measurable changes associated with the disease, including decreased vessel diameter and possible occlusion due to vessel damage and polyurea. We also observed an increase in the blood flow speed in the vein and a decrease in the artery, which could be due to compensation for the dehydration and vessel diameter changes. Functional and metabolic parameters such as hemoglobin oxygen saturation, oxygen extraction fraction, and oxygen consumption rate were also measured, but showed no significant change.

Single-wavelength functional photoacoustic microscopy in biological tissue

Recently, we developed a reflection-mode relaxation photoacoustic microscope, based on saturation intensity, to measure picosecond relaxation times using a nanosecond laser. Here, using the different relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, the oxygen saturation was quantified in vivo with single-wavelength photoacoustic microscopy. All previous functional photoacoustic microscopy measurements required imaging with multiple-laser-wavelength measurements to quantify oxygen saturation. Eliminating the need for multiwavelength measurements removes the influence of spectral properties on oxygenation calculations and improves the portability and cost-effectiveness of functional or molecular photoacoustic microscopy.

In vivo functional photoacoustic microscopy of cutaneous microvasculature in human skin

Microcirculation is an important component of the cardiovascular system and can be used to assess systemic cardiovascular health. Numerous studies have investigated cutaneous microcirculation as an indicator of cardiovascular related diseases. Such research has shown promising results; however, there are many limitations regarding the employed measurement techniques, such as poor depth and spatial resolution and measurement versatility. Here we show the results of functional cutaneous microvascular experiments measured with photoacoustic microscopy, which provides high spatial resolution and multiparameter measurements. In a set of experiments, microvascular networks located in the palms of volunteers were perturbed by periodic ischemic events, and the subsequent hemodynamic response to the stimulus was recorded. Results indicate that during periods of arterial occlusion, the relative oxygen saturation of the capillary vessels decreased below resting levels, and temporarily increased above resting levels immediately following the occlusion. Furthermore, a hyperemic reaction to the occlusions was measured, and the observation agreed well with similar measurements using more conventional imaging techniques. Due to its exceptional capability to functionally image vascular networks with high spatial resolution, photoacoustic microscopy could be a beneficial biomedical tool to assess microvascular functioning and applied to patients with diseases that affect cardiovascular health. © 2011 Society of Photo-Optical Instrumentation Engineers.

Imaging brain hemodynamic changes during rat forepaw electrical stimulation using functional photoacoustic microscopy

The present study reported the development of a novel functional photoacoustic microscopy (fPAM) system for investigating hemodynamic changes in rat cortical vessels associated with electrical forepaw stimulation. Imaging of blood optical absorption by fPAM at multiple appropriately-selected and distinct wavelengths can be used to probe changes in total hemoglobin concentration (HbT, i.e., cerebral blood volume [CBV]) and hemoglobin oxygen saturation (SO 2). Changes in CBV were measured by images acquired at a wavelength of 570 nm ( λ 570), an isosbestic point of the molar extinction spectra of oxy- and deoxy-hemoglobin, whereas SO 2 changes were sensed by pixel-wise normalization of images acquired at λ 560 or λ 600 to those at λ 570. We demonstrated the capacity of the fPAM system to image and quantify significant contralateral changes in both SO 2 and CBV driven by electrical forepaw stimulation. The fPAM system complements existing imaging techniques, with the potential to serve as a favorable tool for explicitly studying brain hemodynamics in animal models.

In vivo imaging of subcutaneous structures using functional photoacoustic microscopy

Functional photoacoustic microscopy (fPAM) is a hybrid technology that permits noninvasive imaging of the optical absorption contrast in subcutaneous biological tissues. fPAM uses a focused ultrasonic transducer to detect high-frequency photoacoustic (PA) signals. Volumetric images of biological tissues can be formed by two-dimensional raster scanning, and functional parameters can be further extracted from spectral measurements. fPAM is safe and applicable to animals as well as humans. This protocol provides guidelines for parameter selection, system alignment, imaging operation, laser safety and data processing for in vivo fPAM. It currently takes approximately 100 min to carry out this protocol, including approximately 50 min for data acquisition using a 10-Hz pulse-repetition-rate laser system. The data acquisition time, however, can be significantly reduced by using a laser system with a higher pulse repetition rate.

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 approximately 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.