Roughly 400,000 children each year are born with port wine stain (PWS) birthmarks, which are skin capillary malformations characterized by a pink-reddish color that develop into purple nodules if left untreated. While PWS birthmarks can be found anywhere on the body and are typically non-life threatening, a large majority (~90%) present on the head and neck regions and can lead to complications including dental abnormalities and may be associated with capillary malformations in other organs such as the eyes (glaucoma), and brain (seizures). Due to the unsightly nature of PWS, people that have them on exposed areas of skin (face, neck, arm or leg regions) can endure immense psychological and social suffering. The current gold standard treatment for PWS in the US is laser irradiation using the pulsed dye laser (PDL). Treatment of PWS with the PDL has limited efficacy; many patients undergo 15 or more laser treatments without seeing full clearance of the PWS, demonstrating the need to investigate alternative therapies.

In my dissertation research, I investigated alternatives to PDL treatment for PWS, including photodynamic therapy (PDT), combined PDT/PDL protocols, and alternative light based treatments. PDT has demonstrated promise towards achieving better treatment outcomes for PWS birthmarks, however, careful selection of treatment parameters is needed to avoid unwanted side effects. I performed preclinical studies of PDT using two photosensitizers, NPe6 and Hemoporfin, to better develop safe treatment parameters. I then investigated a combined PDT/PDL protocol and determined that a synergistic effect exists between the PDT and PDL protocols, illustrating that this combined approach leads to a desired treatment outcome while minimizing side effects. To better understand the PDT treatment mechanisms in skin, I also studied the depth of injury caused by NPe6-mediated PDT in preclinical models and found a direct relationship between depth of vascular injury and light dose administered. Finally, in investigating alternatives to the PDL, I studied the use of intense pulsed light (IPL) and performed a direct comparison study to the PDL. I used computational models to characterize the predicted performance of the IPL and performed experimental studies in preclinical models.

Quantitative characterization of blood flow is important to assess acute physiological health and hemodynamic effects of clinical interventions. A critical need exists for a robust device designed to assess blood flow in biomedical applications. Laser Speckle Imaging (LSI) is a wide-field non-invasive optical technique that enables superficial blood flow quantification. A few potential clinical applications of LSI include assessing blood flow during burn patient triaging, quantifying tissue perfusion in a neonatal intensive care unit, and real-time blood flow mapping during surgery. The blood flow information that LSI provides can be helpful for bedside care. However, since LSI systems are highly sensitive to motion, most LSI studies are typically performed using mounted systems. Widespread use of LSI in the clinic has not occurred, in part, due to the bulky form factor and lack of mobility of these systems.

Our proposed solution to address the limitations of these mounted systems is a handheld LSI device. Handheld LSI would provide clinicians with objective blood flow measurements in a convenient form factor. However, motion artifact during handheld data acquisition can lead to unreliable and inaccurate blood flow values. Attempts have been made to account for motion artifact noise, but they lack an approach to align (co-register) images. Co-registration is a necessary step prior to the common practice of image averaging to improve the signal-to-noise ratio in producing blood flow maps. Our approach to address both motion artifact and image co-registration for handheld LSI was adding a fiducial marker (FM) into our imaging protocol.

We developed a portable, handheld LSI device and a protocol that integrated a FM into the imaging workflow. We automated the processes of sorting frames based on motion artifact and co-registering misaligned images. We compared the performance of the mounted and handheld setups using in vitro flow phantom experiments as a proof-of-concept study. We then demonstrated translation of our imaging protocol into an ongoing in vivo study with a porcine burn wound model.

We attempted to further reduce motion artifact by making additional modifications to the handheld LSI device. The modified LSI device was also validated with in vitro flow phantom experiments and in vivo imaging of vessels within a dorsal skinfold window chamber model.

In addition to the concerns of motion artifacts for clinical imaging, optical property changes in dynamic wounds or developing tissues may cause inaccuracies in the measured blood flow. We addressed this issue by combining LSI and Spatial Frequency Domain Imaging (SFDI), a non-contact imaging modality used to quantify tissue optical properties, to obtain corrected blood flow values. We then showed the potential errors in blood flow values when optical properties are not properly accounted for in the dynamic wounds of a porcine burn wound model.

Collectively, these works demonstrate our attempts at providing a viable alternative for clinical blood flow imaging with our handheld LSI device and imaging protocol and accounting for motion artifact and optical property changes in LSI.

Annually, over 550,000 people in the United States suffer cardiac arrest (CA) and initial survival rates remain low (10-30%). Approximately 85 to 90% of survivors have impaired cognitive function, which leads to decreased quality of life for survivors, increased burden on caregivers, and direct costs of over $6 billion/year. To improve neurological recovery and understand how the brain recovers following CA, a critical need exists to investigate physiological processes that impact the brain. In collaboration with Dr. Yama Akbari’s lab, we developed a multi-modal platform with high temporal (10s of milliseconds) and spatial (~50µm) resolution. The platform combined laser speckle imaging (LSI), arterial blood pressure (ABP), and electroencephalography (EEG) to monitor cerebral blood flow (CBF), peripheral blood pressure, and brain electrophysiology, respectively. We applied this platform to an asphyxial CA and resuscitation preclinical model established in Dr. Akbari’s lab and characterized the hyperdynamic spatiotemporal cerebral dynamics that occur before, during, and after asphyxial CA and resuscitation.

The beginning of neurological recovery post-CA is the moment at which brain electrical activity resumes. We assessed the temporal evolution of CBF prior to this time point, and found two distinct CBF phases. The first phase is a hyperemic (increase in CBF) period that occurs 5-10min after resuscitation, which is followed by a period of prolonged, stabilized hypoperfusion (decrease in CBF). We found that brain electrical activity resumes after the CBF hyperemic period, but before CBF stabilizes hypoperfused. CBF and MAP data were then used to develop an empirical model to predict when brain electrical activity resumes to within 10% accuracy. The link between hemodynamics and the resumption of brain electrical activity, suggests that clinicians may be able to utilize and develop hemodynamic-altering therapeutics that modify the beginning of neurological recovery.

Next, we analyzed pulsatile CBF and femoral ABP waveforms in response to CA and resuscitation. Pulsatile blood flow is important due to its impact on oxygen uptake, vascular tone, and cellular metabolism. Our study revealed that CBF pulsatility is altered significantly from baseline within 2h after resuscitation, but ABP pulsatility changes little. Interestingly, ABP pulsatility and not CBF pulsatility correlates with short-term neurological outcome. These results suggest that simultaneous monitoring of CBF and ABP pulsatility is necessary, and therapeutic modulation of pulsatility post-CA may lead to changes in neurological outcome.

Lastly, we analyzed spatiotemporal CBF changes during three hyperdynamic periods: (1) entering CA, (2) within 5min after resuscitation, and (3) from hyperemia to hypoperfusion (i.e., 5 to 20min after resuscitation). Spreading CBF waves were visualized during each phase. We found that an increase in total CBF prior to the onset of spreading wave (1) was associated with worse neurological outcome, waves (1) and (3) propagated in opposite directions, and waves (2) and (3) resembled a well-known phenomenon called cortical spreading depression. These spatiotemporal characteristics are complex, yet may have clinical importance to neurological outcome.

Collectively, these findings highlight the complex nature of the hyperdynamic cerebral response to CA and resuscitation. The multi-modal platform and results suggest a novel therapeutic approach involving modulation of hemodynamics during hyperdynamic periods of CA may improve neurological outcome.

Port wine stains (PWS) are congenital vascular malformation of the skin that occur in 3-4 infants

per 1000 live births. A major factor that contributes to treatment failures of Port wine stain

(PWS) birthmarks is reperfusion of photocoagulated blood vessels due to wound healing

response. The study of physiological processes that govern the microvasculature plays a major

role in our ability to discover solutions toward enhancement of the current treatments to port

wine stain and many other diseases. The functionality of microvasculature and their response to

external influences is difficult to assess with the traditional analysis techniques such as histology.

The work described in this thesis aims to provide a microvascular optical functional imaging

solution specifically designed for use with the rodent dorsal window chamber models.

Our microvascular imaging system was designed to obtain images with three different imaging

modes: Laser speckle imaging (LSI), Multispectral Imaging/Oxygenation imaging (MSI/O2I)

and Fluorescence imaging (FI). Each of these imaging techniques provides information about the

different aspects of microvessels. LSI is utilized to measure the relative changes in blood flow,

MSI enables computation of hemoglobin saturation maps, and FI highlights biochemical

processes active in a subject. Our analysis showed that the developed imaging system that

combines different imaging techniques could be used as a valuable tool to assess the behavior of

microvessels over time in response to external factors.

Microscopy is a fundamental tool in scientific research, allowing the visualization ofstructures not visible to the naked eye. However, imaging thick tissue samples presents challenges due to light scattering from mismatched refractive indices. Tissue clearing techniques solve these issues by rendering tissues transparent, enhancing image quality and contrast-to-background ratios. Despite benefits, common methods can result in tissue deformation, long processing times, and fluorescence quenching. To address these issues, two advanced techniques have been developed: immunolabeling-enabled solvent-cleared organs (iDISCO) and the Fast Optical Clearing Method (FOCM). iDISCO is highly effective in large-scale tissue clearing, ideal for comprehensive studies, but uses hazardous chemicals and requires days to clear tissues. In contrast, FOCM offers a safer, more rapid alternative, achieving tissue transparency in as little as five minutes while maintaining vessel intensity and preserving fluorescence. This study aims to compare the efficacy of FOCM to iDISCO in enhancing imaging quality, focusing on vessel intensity and contrast-to-background ratios in one-millimeter-thick mouse brain samples. By comparing one-millimeter-thick mouse brain samples, the study found that FOCM maintained vessel intensity and contrast-tobackground ratios comparable to iDISCO. FOCM was also tested at 5, 60, and 48 hours, demonstrating increased vessel intensity and contrast over time, with 48 hours providing optimal results. This makes FOCM particularly useful for time-sensitive analyses while maintaining image quality