Sean M. White

Wide-field functional imaging of blood flow and hemoglobin oxygen saturation in the rodent dorsal window chamber

The rodent dorsal window chamber is a widely used in vivo model of the microvasculature. The model consists of a 1cm region of exposed microvasculature in the rodent dorsal skin that is immobilized by surgically implanted titanium frames, allowing the skin microvasculature to be visualized. We describe a detailed protocol for surgical implantation of the dorsal window chamber which enables researchers to perform the window chamber implantation surgery. We further describe subsequent wide-field functional imaging of the chamber to obtain hemodynamic information in the form of blood oxygenation and blood flow on a cm size region of interest. Optical imaging techniques, such as intravital microscopy, have been applied extensively to the dorsal window chamber to study microvascular-related disease and conditions. Due to the limited field of view of intravital microscopy, detailed hemodynamic information typically is acquired from small regions of interest, typically on the order of hundreds of μm. The wide-field imaging techniques described herein complement intravital microscopy, allowing researchers to obtain hemodynamic information at both microscopic and macroscopic spatial scales. Compared with intravital microscopy, wide-field functional imaging requires simple instrumentation, is inexpensive, and can give detailed metabolic information over a wide field of view.

Automated computation of functional vascular density using laser speckle imaging in a rodent window chamber model.

We report a methodology for computing functional vascular density within a rodent dorsal window chamber model based on long-exposure laser speckle imaging (LSI). This technique relies on the presence of flow to create detailed vasculature maps. Employing this contrast mechanism is not possible using conventional imaging methods. Additionally, a freeware algorithm for computing functional vascular density (FVD) from images acquired using long-exposure LSI is also described to facilitate ease in adopting this method. We demonstrate that together these tools can be used to compute FVD nearly twelve times faster than manual computation, yet with comparable accuracy.

Noninvasive evaluation of the vascular response to transplantation of alginate encapsulated islets using the dorsal skin-fold model

Alginate encapsulation reduces the risk of transplant rejection by evading immune-mediated cell injury and rejection; however, poor vascular perfusion results in graft failure. Since existing imaging models are incapable of quantifying the vascular response to biomaterial implants after transplantation, in this study, we demonstrate the use of in vivo laser speckle imaging (LSI) and wide-field functional imaging (WiFI) to monitor the microvascular environment surrounding biomaterial implants. The vascular response to two islet-containing biomaterial encapsulation devices, alginate microcapsules and a high-guluronate alginate sheet, was studied and compared after implantation into the mouse dorsal window chamber (N = 4 per implant group). Images obtained over a 14-day period using LSI and WiFI were analyzed using algorithms to quantify blood flow, hemoglobin oxygen saturation and vascular density. Using our method, we were able to monitor the changes in the peri-implant microvasculature noninvasively without the use of fluorescent dyes. Significant changes in blood flow, hemoglobin oxygen saturation and vascular density were noted as early as the first week post-transplant. The dorsal window chamber model enables comparison of host responses to transplanted biomaterials. Future experiments will study the effect of changes in alginate composition on the vascular and immune responses.

Implanted Cell-Dense Prevascularized Tissues Develop Functional Vasculature That Supports Reoxygenation After Thrombosis

Achieving adequate vascularization within implanted engineered tissues is a significant obstacle to maintaining viability and functionality. In vitro prevascularization of engineered tissues has been explored as a potential solution to this challenge. The traditional paradigm of in vitro prevascularization is to implant an engineered tissue with a preformed vascular network that is perfused after anastomosis with the host circulation. We investigated the efficacy of this strategy by implanting cell-dense prevascularized tissues created via cell-mediated contraction and composed of collagen and a collagen-fibrin mixture into dorsal window chambers surgically prepared on immunocompromised mice. We found that host-implant anastomosis takes place in 2-6 days and that perfusion of vessels within the implants is subsequently restricted by thrombosis. However, by day 7, a functional vascular network composed of host and implant vessels developed. Prevascularization enhanced intra-implant pO2 significantly as early as 2 days postimplantation, reaching a maximum of 55 mmHg by day 8, which was significantly greater than the maximum within cellularized control tissues (18 mmHg). By day 14, collagen tissues supported ∼ 0.51 × 10(9) implanted and host-derived cells per mL. Our findings elucidate key features of in vitro prevascularization that can be used toward the design of larger and more functionally complex engineered tissues.

Enzymatically activated near infrared nanoprobes based on amphiphilic block copolymers for optical detection of cancer

Nanotechnology offers the possibility of creating multi‐functional structures that can provide solutions for biomedical problems. The nanoprobes herein described are an example of such structures, where nano‐scaled particles have been designed to provide high specificity and contrast potential for optical detection of cancer. Specifically, enzymatically activated fluorescent nanoprobes (EANPs) were synthesized as cancer‐specific contrast agents for optical imaging.

The Role of Laser Speckle Imaging in Port-Wine Stain Research: Recent Advances and Opportunities

Here, we review our current knowledge on the etiology and treatment of port-wine stain (PWS) birthmarks. Current treatment options have significant limitations in terms of efficacy. With the combination of 1) a suitable preclinical microvascular model, 2) laser speckle imaging (LSI) to evaluate blood-flow dynamics, and 3) a longitudinal experimental design, rapid preclinical assessment of new phototherapies can be translated from the lab to the clinic. The combination of photodynamic therapy (PDT) and pulsed-dye laser (PDL) irradiation achieves a synergistic effect that reduces the required radiant exposures of the individual phototherapies to achieve persistent vascular shutdown. PDL combined with antiangiogenic agents is a promising strategy to achieve persistent vascular shutdown by preventing reformation and reperfusion of photocoagulated blood vessels. Integration of LSI into the clinical workflow may lead to surgical image guidance that maximizes acute photocoagulation, which is expected to improve PWS therapeutic outcome. Continued integration of noninvasive optical imaging technologies and biochemical analysis collectively are expected to lead to more robust treatment strategies.

Blood flow quantification of biopsied skin lesions using a laser speckle imaging dermatoscope (Conference Presentation)

Dermatoscopes are commonly utilized by medical professionals for the qualitative visual inspection of skin lesions. While automated image processing techniques and varied illumination strategies can aid in structural analysis of lesions, robust quantification of functional information is largely unknown. To address this knowledge gap, we have developed a compact, handheld dermatoscope that enables real-time blood flow measurements of skin using coherent illumination and laser speckle imaging (LSI). A second color camera attached to the dermatoscope helps with the simultaneous real-time observation of the skin lesions and allows the user to acquire and save color images via a custom Graphical User Interface. In-vitro characterization utilizing a blood flow phantom demonstrated that the dermatoscope is capable of quantifying changes in blood flow across a physiologically relevant range even when used in a handheld manner with ambient lighting. We also demonstrated that the dermatoscope can quantify blood flow in skin lesions in human subjects and that significant differences in blood flow are present among lesion types. There was significantly increased blood flow relative to the surrounding skin in cherry angiomas compared to solar lentigos (p<0.05), which was expected based on the vascular and pigment compositions of the two lesion types. Furthermore, we have compared blood flow maps collected from potentially cancerous lesions prior to histological analysis to determine whether blood flow measurements can help in the diagnosis of benign and malignant skin lesions. Information provided by the LSI dermatoscope may help with earlier and more accurate diagnoses of pigmented skin lesions.

Histologic changes associated with talaporfin sodium‐mediated photodynamic therapy in rat skin

Alternative treatments are needed to achieve consistent and more complete port wine stain (PWS) removal, especially in darker skin types; photodynamic therapy (PDT) is a promising alternative treatment. To this end, we previously reported on Talaporfin Sodium (TS)‐mediated PDT. It is essential to understand treatment tissue effects to design a protocol that will achieve selective vascular injury without ulceration and scarring. The objective of this work is to assess skin changes associated with TS‐mediated PDT with clinically relevant treatment parameters.

Design and evaluation of a miniature laser speckle imaging device to assess gingival health

Abstract. Current methods used to assess gingivitis are qualitative and subjective. We hypothesized that gingival perfusion measurements could provide a quantitative metric of disease severity. We constructed a compact laser speckle imaging (LSI) system that could be mounted in custom-made oral molds. Rigid fixation of the LSI system in the oral cavity enabled measurement of blood flow in the gingiva. In vitro validation performed in controlled flow phantoms demonstrated that the compact LSI system had comparable accuracy and linearity compared to a conventional bench-top LSI setup. In vivo validation demonstrated that the compact LSI system was capable of measuring expected blood flow dynamics during a standard postocclusive reactive hyperemia and that the compact LSI system could be used to measure gingival blood flow repeatedly without significant variation in measured blood flow values (p<0.05). Finally, compact LSI system measurements were collected from the interdental papilla of nine subjects and compared to a clinical assessment of gingival bleeding on probing. A statistically significant correlation (ρ=0.53; p<0.005) was found between these variables, indicating that quantitative gingival perfusion measurements performed using our system may aid in the diagnosis and prognosis of periodontal disease.