Austin J. Moy

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.

Optical Histology: A Method to Visualize Microvasculature in Thick Tissue Sections of Mouse Brain

Background The microvasculature is the network of blood vessels involved in delivering nutrients and gases necessary for tissue survival. Study of the microvasculature often involves immunohistological methods. While useful for visualizing microvasculature at the µm scale in specific regions of interest, immunohistology is not well suited to visualize the global microvascular architecture in an organ. Hence, use of immunohistology precludes visualization of the entire microvasculature of an organ, and thus impedes study of global changes in the microvasculature that occur in concert with changes in tissue due to various disease states. Therefore, there is a critical need for a simple, relatively rapid technique that will facilitate visualization of the microvascular network of an entire tissue. Methodology/Principal Findings The systemic vasculature of a mouse is stained with the fluorescent lipophilic dye DiI using a method called “vessel painting”. The brain, or other organ of interest, is harvested and fixed in 4% paraformaldehyde. The organ is then sliced into 1 mm sections and optically cleared, or made transparent, using FocusClear, a proprietary optical clearing agent. After optical clearing, the DiI-labeled tissue microvasculature is imaged using confocal fluorescence microscopy and adjacent image stacks tiled together to produce a depth-encoded map of the microvasculature in the tissue slice. We demonstrated that the use of optical clearing enhances both the tissue imaging depth and the estimate of the vascular density. Using our “optical histology” technique, we visualized microvasculature in the mouse brain to a depth of 850 µm. Conclusions/Significance Presented here are maps of the microvasculature in 1 mm thick slices of mouse brain. Using combined optical clearing and optical imaging techniques, we devised a methodology to enhance the visualization of the microvasculature in thick tissues. We believe this technique could potentially be used to generate a three-dimensional map of the microvasculature in an entire organ.

High-resolution visualization of mouse cardiac microvasculature using optical histology

Cardiovascular disease typically is associated with dysfunction of the coronary vasculature and microvasculature. The study of cardiovascular disease typically involves imaging of the large coronary vessels and quantification of cardiac blood perfusion. These methods, however, are not well suited for imaging of the cardiac microvasculature. We used the optical histology method, which combines chemical optical clearing and optical imaging, to create high-resolution, wide-field maps of the cardiac microvasculature in ventral slices of mouse heart. We have demonstrated the ability of the optical histology method to enable wide-field visualization of the cardiac microvasculature in high-resolution and anticipate that optical histology may have significant impact in studying cardiovascular disease.

Combinatorial immunotherapy and nanoparticle mediated hyperthermia

Immune checkpoint therapy has become the first widely adopted immunotherapy for patients with late stage malignant melanoma, with potential for a wide range of cancers. While some patients can experience long term disease remission, this is limited only to a subset of patients and tumor types. The path forward to expand this therapy to more patients and tumor types is currently thought to be combinatorial treatments, the combination of immunotherapy with other treatments. In this review, the combinatorial approach of immune checkpoint therapy combined with nanoparticle-assisted localized hyperthermia is discussed, starting with an overview of the different nanoparticle hyperthermia approaches in development, an overview of the state of immune checkpoint therapy, recent reports of immune checkpoint therapy and nanoparticle-assisted hyperthermia in a combinatorial approach, and finally a discussion of future research topics and areas to be explored in this new combinatorial approach to cancer treatment.

Conductive polymer-based nanoparticles for laser-mediated photothermal ablation of cancer: synthesis, characterization, and in vitro evaluation

Laser-mediated photothermal ablation of cancer cells aided by photothermal agents is a promising strategy for localized, externally controlled cancer treatment. We report the synthesis, characterization, and in vitro evaluation of conductive polymeric nanoparticles (CPNPs) of poly(diethyl-4,4′-{[2,5-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1,4-phenylene] bis(oxy)}dibutanoate) (P1) and poly(3,4-ethylenedioxythiophene) (PEDOT) stabilized with 4-dodecylbenzenesulfonic acid and poly(4-styrenesulfonic acid-co-maleic acid) as photothermal ablation agents. The nanoparticles were prepared by oxidative-emulsion polymerization, yielding stable aqueous suspensions of spherical particles of <100 nm diameter as determined by dynamic light scattering and electron microscopy. Both types of nanoparticles show strong absorption of light in the near infrared region, with absorption peaks at 780 nm for P1 and 750 nm for PEDOT, as well as high photothermal conversion efficiencies (~50%), that is higher than commercially available gold-based photothermal ablation agents. The nanoparticles show significant photostability as determined by their ability to achieve consistent temperatures and to maintain their morphology upon repeated cycles of laser irradiation. In vitro studies in MDA-MB-231 breast cancer cells demonstrate the cytocompatibility of the CPNPs and their ability to mediate complete cancer cell ablation upon irradiation with an 808-nm laser, thereby establishing the potential of these systems as agents for laser-induced photothermal therapy.

Optical properties of mouse brain tissue after optical clearing with FocusClear

Abstract. Fluorescence microscopy is commonly used to investigate disease progression in biological tissues. Biological tissues, however, are strongly scattering in the visible wavelengths, limiting the application of fluorescence microscopy to superficial (<200  μm) regions. Optical clearing, which involves incubation of the tissue in a chemical bath, reduces the optical scattering in tissue, resulting in increased tissue transparency and optical imaging depth. The goal of this study was to determine the time- and wavelength-resolved dynamics of the optical scattering properties of rodent brain after optical clearing with FocusClear™. Light transmittance and reflectance of 1-mm mouse brain sections were measured using an integrating sphere before and after optical clearing and the inverse adding doubling algorithm used to determine tissue optical scattering. The degree of optical clearing was quantified by calculating the optical clearing potential (OCP), and the effects of differing OCP were demonstrated using the optical histology method, which combines tissue optical clearing with optical imaging to visualize the microvasculature. We observed increased tissue transparency with longer optical clearing time and an analogous increase in OCP. Furthermore, OCP did not vary substantially between 400 and 1000 nm for increasing optical clearing durations, suggesting that optical histology can improve ex vivo visualization of several fluorescent probes.

Talaporfin sodium-mediated photodynamic therapy alone and in combination with pulsed dye laser on cutaneous vasculature

Currently, standard treatment for port wine stain (PWS) birthmarks in the United States involves use of lasers or intense pulsed light to photocoagulate selectively the abnormal vasculature. With photothermal therapy, PWS often become lighter, but patients must undergo many treatments (15–20 are not uncommon; Koster et al, 2001). Furthermore, treatment of skin types IV–VI is difficult due to absorption of light by overlying epidermal melanin, limiting treatment safety and efficacy. Photodynamic therapy (PDT), an alternative treatment option, involves optical excitation of an exogenous photosensitizer and subsequent energy transfer from the photosensitizer to oxygen to create cytotoxic singlet oxygen (Gorman et al, 2006). Excitation of photosensitizers localized primarily within the intravascular compartment, enables targeted vascular destruction. Treatment can be effective but is associated with prolonged photosensitivity and substantial scarring risk (Lu et al, 2010). Talaporfin sodium (TS) is a photosensitizer with proven selective vascular effects in preclinical studies, an acceptable photosensitivity period of 5–7 days, and good safety data (Akimoto et al, 2012; Bromley et al 2011; Kujundzic et al 2007). Previously, we determined that the characteristic radiant exposure required for persistent vascular shutdown (RE50/7 value) for TS-mediated PDT, using a custom-built LED array (664nm, FWHM = 20nm), was 85J/cm2 (Moy et al, 2012). Based on these and other published data (Channual et al, 2008; Smith et al, 2006; Tournas et al, 2009), we hypothesized that dual phototherapy treatment with TS-mediated PDT and ensuing PDL therapy, will achieve persistent vascular shutdown with otherwise sub-therapeutic radiant exposures of PDT and PDL. To test this hypothesis, we performed studies to determine the RE50/7 to achieve persistent vascular shutdown with PDL irradiation and the associated RE50/7 values for dual phototherapy. We postulate that lower radiant exposures can be used for both TS-mediated PDT and ensuing PDL, minimizing adverse effects, allowing treatment of all skin types and potentially achieving enhanced treatment efficacy, compared to either alone. Utilizing a UC Irvine Institutional Animal Care and Use Committee approved protocol, we installed dorsal window chambers (Moy et al, 2011) on adult C3H mice (25–30g, n=38) anesthetized with isoflurane. For PDT, we utilized a custom-built light emitting diode array centered at 664-nm excitation (FWHM=20nm). For TS-mediated PDT, we reconstituted TS (Light Sciences Oncology; Bellevue, Washington) using sterile saline to form a solution of 25 mg/mL. We injected TS (5 mg/kg) into the bloodstream via retro-orbital injection and began PDT immediately afterwards (irradiance 100 mW/cm2; radiant exposure 0–260 J/cm2). For PDL irradiation, we used a clinical 595-nm laser (Vbeam Perfecta, Candela Corporation, Wayland, MA; 10 mm diameter spot size, 1.5 ms pulse duration, radiant exposure 3.25–10.00 J/cm2). We randomized experiment order. To test the hypothesis that the dual therapy protocol enables persistent vascular shutdown with lower radiant exposures of either PDT or PDL irradiation, we restricted our study to radiant exposure values of PDT (20–60 J/cm2) and PDL (4–6 J/cm2) that were below the associated RE50/7 values for either treatment alone. We performed PDL irradiation within 5 s after PDT. To monitor blood-flow dynamics, we used Laser Speckle Imaging (LSI) (Moy et al, 2011). We used an experimental design based on dose-response analysis (Moy et al, 2012). We performed 19 experiments to establish a dose-response curve for PDL and 19 experiments for PDT+PDL. We collected raw speckle images before and at time points during the ensuing week (Choi et al, 2008). Five of the authors (BC, WJM, KMK, BSL, and JJM) independently reviewed the SFI images collected on Day 7 and graded them as “0” (no persistent vascular shutdown) or “1” (persistent vascular shutdown achieved). Prism (version 5.0d, GraphPad Software, San Diego, CA) was used to estimate the RE50/7 for each study. We used a F-test to compare the log (RE50/7) values determined from PDT (85J/cm2 from Moy et al., 2012) and PDT+PDL. Our null hypothesis was that the RE50/7 values for the two studies do not differ in a statistically significant manner. We observed three dose-dependent responses: 1) minimal acute change in blood flow and no persistent vascular shutdown (Figure 1A); 2) marked acute change in blood flow, followed by partial-to-full recovery of blood flow and no persistent vascular shutdown (Figure 1B); and 3) marked acute or delayed reduction in blood flow, followed by complete vascular shutdown at Day 7 (Figure 1C). With application of dose-response methodology, we estimated a RE50/7 of 7.1J/cm2 required to induce persistent vascular shutdown with PDL irradiation (Figure 1D). With PDT+PDL, the characteristic PDT radiant exposure required to achieve persistent vascular shutdown, decreased from 85 to 45J/cm2 (Figure 1E). This difference in PDT RE50/7 was found to be statistically significant (p=0.0002). Figure 1 The combination of TS-mediated PDT and PDL irradiation leads to a significant reduction in the characteristic PDT radiant exposure required to achieve persistent vascular shutdown We evaluated the degree of synergy between PDT and PDL vascular effects with dual phototherapy (Madsen et al., 2002): α=fPDTfPDLfPDT+PDL (1) where fPDT and fPDL are the fractions of single phototherapy experiments and fPDT+PDL is the fraction of combined experiments, which do not induce persistent vascular shutdown. An additive (or absence of any) effect is indicated by α=1, α>1 indicates a synergistic effect, and α<1 indicates an antagonistic effect. Our data (Table 1) suggest the synergistic nature (α=2.7) of PDT+PDL. Collectively, our results reveal that PDT+PDL reduces the PDT light dose required to achieve persistent vascular shutdown, even at low PDL radiant exposures. We hypothesize that TS-mediated PDT enhances persistent vascular shutdown achieved with ensuing PDL therapy, primarily via endothelial cell damage (Mitra and Foster 2008); mechanistic studies currently are underway. Table 1 Summary of observations of persistent vascular shutdown for experiments in which the PDT radiant exposure was 20–60J/cm2 and/or the PDL radiant exposure was 4–6J/cm2. The PDT data is taken from Moy et al. (2012). Based on these data and ... Dual phototherapy represents a potential new approach for more effective treatment of PWS birthmarks. We have initiated an Investigational Review Board approved trial to evaluate intravenously administered TS/664 nm laser light mediated dual phototherapy for PWS treatment. Treatment has been painless and notable lesion lightening has been achieved with both PDT and PDT+PDL in a single session. Patients are photosensitive for 5–7 days post-procedure and for the first 72 hours must remain indoors with lights dimmed. Completion of this study will determine if lesion lightening is greater with dual phototherapy than PDL alone. It is our intent that this combined low energy dual phototherapy will offer clinicians and patients of all skin types improved lesion lightening in fewer treatments.

Raman active components of skin cancer

Raman spectroscopy (RS) has shown great potential in noninvasive cancer screening. Statistically based algorithms, such as principal component analysis, are commonly employed to provide tissue classification; however, they are difficult to relate to the chemical and morphological basis of the spectroscopic features and underlying disease. As a result, we propose the first Raman biophysical model applied to in vivo skin cancer screening data. We expand upon previous models by utilizing in situ skin constituents as the building blocks, and validate the model using previous clinical screening data collected from a Raman optical fiber probe. We built an 830nm confocal Raman microscope integrated with a confocal laser-scanning microscope. Raman imaging was performed on skin sections spanning various disease states, and multivariate curve resolution (MCR) analysis was used to resolve the Raman spectra of individual in situ skin constituents. The basis spectra of the most relevant skin constituents were combined linearly to fit in vivo human skin spectra. Our results suggest collagen, elastin, keratin, cell nucleus, triolein, ceramide, melanin and water are the most important model components. We make available for download (see supplemental information) a database of Raman spectra for these eight components for others to use as a reference. Our model reveals the biochemical and structural makeup of normal, nonmelanoma and melanoma skin cancers, and precancers and paves the way for future development of this approach to noninvasive skin cancer diagnosis.

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.

Color structured light imaging of skin

We illustrate wide-field imaging of skin using a structured light (SL) approach that highlights the contrast from superficial tissue scattering. Setting the spatial frequency of the SL in a regime that limits the penetration depth effectively gates the image for photons that originate from the skin surface. Further, rendering the SL images in a color format provides an intuitive format for viewing skin pathologies. We demonstrate this approach in skin pathologies using a custom-built handheld SL imaging system.