Jeeun Kang

Prostate-specific membrane antigen-targeted photoacoustic imaging of prostate cancer in vivo

A sensitive, noninvasive method to detect localized prostate cancer, particularly for early detection and repetitive study in patients undergoing active surveillance, remains an unmet need. Here, we propose a molecular photoacoustic (PA) imaging approach by targeting the prostate-specific membrane antigen (PSMA), which is over-expressed in the vast majority of prostate cancers. We performed spectroscopic PA imaging in an experimental model of prostate cancer, namely, in immunocompromised mice bearing PSMA+ (PC3 PIP) and PSMA- (PC3 flu) tumors through administration of the known PSMA-targeted fluorescence agent, YC-27. Differences in contrast between PSMA+ and isogenic control tumors were observed upon PA imaging, with PSMA+ tumors showing higher contrast in average of 66.07-fold with 5 mice at the 24-hour postinjection time points. These results were corroborated using standard near-infrared fluorescence imaging with YC-27, and the squared correlation between PA and fluorescence intensities was 0.89. Spectroscopic PA imaging is a new molecular imaging modality with sufficient sensitivity for targeting PSMA in vivo, demonstrating the potential applications for other saturable targets relevant to cancer and other disorders.

Validation of Noninvasive Photoacoustic Measurements of Sagittal Sinus Oxyhemoglobin Saturation in Hypoxic Neonatal Piglets

We hypothesize that noninvasive photoacoustic imaging can accurately measure cerebral venous oxyhemoglobin saturation (So2) in a neonatal model of hypoxia-ischemia. In neonatal piglets, which have a skull thickness comparable to that of human neonates, we compared the photoacoustic measurement of sagittal sinus So2 against that measured directly by blood sampling over a wide range of conditions. Systemic hypoxia was produced by decreasing inspired oxygen stepwise (i.e., 100, 21, 19, 17, 15, 14, 13, 12, 11, and 10%) with and without unilateral or bilateral ligation of the common carotid arteries to enhance hypoxia-ischemia. Transcranial photoacoustic sensing enabled us to detect changes in sagittal sinus O2 saturation throughout the tested range of 5-80% without physiologically relevant bias. Despite lower cortical perfusion and higher oxygen extraction in groups with carotid occlusion at equivalent inspired oxygen, photoacoustic measurements successfully provided a robust linear correlation that approached the line of identity with direct blood sample measurements. Receiver-operating characteristic analysis for discriminating So2 <30% showed an area under the curve of 0.84 for the pooled group data, and 0.87, 0.91, and 0.92 for hypoxia alone, hypoxia plus unilateral occlusion, and hypoxia plus bilateral occlusion subgroups, respectively. The detection precision in this critical range was confirmed with sensitivity (87.0%), specificity (86.5%), accuracy (86.8%), positive predictive value (90.5%), and negative predictive value (81.8%) in the combined dataset. These results validate the capability of photoacoustic sensing technology to accurately monitor sagittal sinus So2 noninvasively over a wide range and support its use for early detection of neonatal hypoxia-ischemia. NEW & NOTEWORTHY We present data to validate the noninvasive photoacoustic measurement of sagittal sinus oxyhemoglobin saturation. In particular, this paper demonstrates the robustness of this methodology during a wide range of hemodynamic and physiological changes induced by the stepwise decrease of fractional inspired oxygen to produce hypoxia and by unilateral and bilateral ligation of the common carotid arteries preceding hypoxia to produce hypoxia-ischemia. This technique may be useful for diagnosing risk of neonatal hypoxic-ischemic encephalopathy.

Recording membrane potential changes through photoacoustic voltage sensitive dye

Monitoring of the membrane potential is possible using voltage sensitive dyes (VSD), where fluorescence intensity changes in response to neuronal electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. In contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near infrared light excitation and ultrasound detection. In this work, we develop the theoretical concept whereby the voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. Based on this concept, we synthesized a novel near infrared photoacoustic VSD (PA-VSD) whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. With a 3-9 μM VSD concentration, we measured a PA signal increase in the range of 5.3 % to 18.1 %, and observed a corresponding signal reduction in fluorescence emission of 30.0 % to 48.7 %. A theoretical model successfully accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate the voltage sensing capability of the dye, but also indicate the necessity of considering both fluorescence and absorbance spectral sensitivities in order to optimize the characteristics of improved photoacoustic probes. Together, our results demonstrate photoacoustic sensing as a potential new modality for sub-second recording and external imaging of electrophysiological and neurochemical events in the brain.

Toward high-speed transcranial photoacoustic imaging using compact near-infrared pulsed LED illumination system

Quantification of brain function is a significant milestone towards understanding of the underlying workings of the brain. Photoacoustic (PA) imaging is the emerging brain sensing modality by which the molecular light absorptive contrast can be non-invasively quantified from deep-lying tissue (~several cm). In this BRAIN initiative effort, we propose high-speed transcranial PA imaging using a novel, compact pulsed LED illumination system (Prexion Inc., Japan) with 200-uJ pulse energy for 75-ns duration, and pulse repetition frequency (PRF) up to 4kHz at near-infrared (NIR) wavelengths of 690-nm and 850-nm switchable in real-time. To validate the efficacy of the proposed system, preliminary ex vivo experiments were conducted with mice skull and human temporal bone, which included vessel-mimicking tubes filled with 10% Indian Ink solution and light absorptive rubber material, respectively. The results indicated that significant PA contrast, 150% signal-to-noise ratio (SNR), can be achieved through the mice skull only with 64 subsequent frame averaging. The minimal number of frames for averaging required was only 16 to generate signal above background noise, leading to 250 Hz frame rate in the strictest temporal frame separation. Furthermore, distinguishable PA contrast was achieved with human temporal bone with 64-frame averaging. Overall, the preliminary results indicate that the LED illumination system can be a cost-effective solution for high-speed PA brain imaging in preclinical and clinical applications, compared to expansive and bulky Nd:YAG laser systems commonly used in PA imaging.

Listening to membrane potential: photoacoustic voltage-sensitive dye recording

Abstract. Voltage-sensitive dyes (VSDs) are designed to monitor membrane potential by detecting fluorescence changes in response to neuronal or muscle electrical activity. However, fluorescence imaging is limited by depth of penetration and high scattering losses, which leads to low sensitivity in vivo systems for external detection. By contrast, photoacoustic (PA) imaging, an emerging modality, is capable of deep tissue, noninvasive imaging by combining near-infrared light excitation and ultrasound detection. Here, we show that voltage-dependent quenching of dye fluorescence leads to a reciprocal enhancement of PA intensity. We synthesized a near-infrared photoacoustic VSD (PA-VSD), whose PA intensity change is sensitive to membrane potential. In the polarized state, this cyanine-based probe enhances PA intensity while decreasing fluorescence output in a lipid vesicle membrane model. A theoretical model accounts for how the experimental PA intensity change depends on fluorescence and absorbance properties of the dye. These results not only demonstrate PA voltage sensing but also emphasize the interplay of both fluorescence and absorbance properties in the design of optimized PA probes. Together, our results demonstrate PA sensing as a potential new modality for recording and external imaging of electrophysiological and neurochemical events in the brain.

Transcranial photoacoustic imaging of NMDA-evoked focal circuit dynamics in rat forebrain

Transcranial photoacoustic (PA) voltage-sensitive dye (VSD) imaging promises to overcome current temporal and spatial limitations of functional neuroimaging. The technique previously distinguished global seizure activity from control neural activity in groups of rats. To validate the focal specificity of transcranial PA with VSD (IR780) imaging in vivo, we now present proofs-of-concept that the results differentiate between N-methyl-D-aspartate (NMDA) evoked neural activities in two distinct circuits with differential response profiles, i.e., sensorimotor cortex and hippocampus. Concurrent quantitative EEG (qEEG) recorded real time circuit dynamics. We hypothesized that NMDA evoked focal glutamate release in both circuits would correlate with increases of location-specific PA signals and time-specific EEG signals. To test the hypothesis in hippocampus, we infused 0.3, 1, and 3 mM NMDA at 2 μl/min over 60 min via an implanted microdialysis probe. The dialysate samples collected every 20 minutes during the infusion were analyzed for focal changes in extracellular glutamate release and quantified by high-performance liquid chromatography (HPLC). Microdialysis at 0.3, 1, and 3 mM showed dose-dependent increases of glutamate (39%, 200% and 670 %, respectively) in hippocampus. We recorded dose-dependent increases of the PA signal with 0.3 and 3 mM NMDA infusion as predicted. Quantitative EEG at 3 mM NMDA infusion confirmed induction of focal seizure activity. Transcranial PA VSD imaging provided up to 301±42% of fractional neural activity index change measured at the contralateral side of the microdialysis probe with 3 mM NMDA infusion, compared to the decrease of −24±34% with 0.3 mM NMDA infusion. This graded response suggests dentate gyrus (DG) gatekeeping in hippocampus. In sensorimotor cortex, intracerebral infusion of NMDA at 0.3 mM elicited the predicted increase of glutamate release, with a decrease of qEEG spectral power. Transcranial PA VSD imaging had - 13±37% of fractional neural activity index change during the 20 min of 0.3 mM NMDA infusion, consistent with surround suppression of depolarization as shown by qEEG of motor cortex. We conclude that transcranial PA VSD imaging distinguished relatively greater cortical inhibition and threshold-level hippocampal focal seizure activity that both correlated with activities reported by qEEG signals. The results show this emerging technology to be an innovative and significant advance of functional neuroimaging.

In vivo photoacoustic quantification of brain tissue oxygenation for neonatal piglet graded ischemia model using microsphere administration

Perinatal arterial ischemic stroke can result in long-term deficits in motor, cognitive, attention, and executive functions as well as persistent seizures. Rapid diagnosis is critical for newborns who suffer a focal ischemic stroke in order to distinguish prenatal vs. postnatal stroke, and to differentiate perinatal stroke from global hypoxic ischemia (HI). In this study, we present photoacoustic (PA) quantification for brain tissue oxygenation on an in vivo focal stroke model based on a newborn piglet.

Real-time intra-operative guidance using combined photoacoustic and pulsed fluorescence imaging for robot-assisted surgical operation

Multi-modal interface in medicine has been of interest as it extends clinical vision and diagnostic to a higher dimension. In this paper, we develop a real-time intra-operative guidance for robot-assisted surgical operation using a dual photoacoustic (PA) and fluorescence (FL) imaging based on a common pulsed laser and commercial ultrasound array transducer.

Real-time recording of neuronal voltage membrane variation during seizure using transcranial photoacoustic voltage-sensitive dye imaging

A great need exists to non-invasively quantify the neurotransmitter activity in real-time to build a comprehensive functional map of a brain. In this paper, we present real-time recording of neuronal membrane potential change in vivo using transcranial photoacoustic (PA) voltage-sensitive dye (VSD) imaging on a rat in seizure.

Transcranial real-time in vivo recording of electrophysiological neural activity in the rodent brain with near-infrared photoacoustic voltage-sensitive dye imaging

Minimally-invasive monitoring of electrophysiological neural activities in real-time—that would enable quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET—presents a very challenging yet significant task for neuroimaging. We present in vivo proof-of-concept results of transcranial photoacoustic (PA) imaging of chemoconvulsant seizure activity in the rat brain. The framework involves use of a fluorescence quenching-based near-infrared voltage-sensitive dye (VSD) delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling. Using normalized time-frequency analysis on temporal PA sequences, the neural activity in the seizure group was distinguished from those of the control groups. Electroencephalogram (EEG) recording confirmed the changes of severity and frequency of brain activities, induced by chemoconvulsant seizures of the rat brain. The findings demonstrate that PA imaging of fluorescence quenching-based VSD is a promising tool for in vivo recording of deep brain activities in the rat brain, thus excluding the need of invasive craniotomy.Abstract: Minimally-invasive monitoring of electrophysiological neural activities in real-time—that enables quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET—presents a very challenging yet significant task for neuroimaging. In this paper, we present proof-of-concept in vivo functional PA (fPA) imaging of chemoconvulsant rat seizure model with intact scalp using a fluorescence quenching-based cyanine voltage-sensitive dye (VSD) characterized by a lipid vesicle model mimicking different levels of membrane potential variation. The framework also involves use of a near-infrared VSD delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling. Using normalized time-frequency analysis on temporal PA sequences, the neural activity in the seizure group was distinguished from those of the control groups. Electroencephalogram (EEG) recording confirmed the changes of severity and frequency of brain activities, induced by chemoconvulsant seizures of the rat brain. The findings demonstrate that fPA imaging of fluorescence quenching-based VSD is a promising tool for in vivo recording of deep brain activities in the rat brain, thus excluding the need of invasive craniotomy.