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Hamster cheek pouches were exposed to 2-chloroethyl ethyl sulfide [CEES, half-mustard gas (HMG)] at a concentration of 0.4, 2.0, or 5.0 mg/ml for 1 or 5 min. Twenty-four hours post-HMG exposure, tissue damage was assessed by both stereomicrography and optical coherence tomography (OCT). Damage that was not visible on gross visual examination was apparent in the OCT images. Tissue changes were found to be dependent on both HMG concentration and exposure time. The submucosal and muscle layers of the cheek pouch tissue showed the greatest amount of structural alteration. Routine light microscope histology was performed to confirm the OCT observations.

A preliminary study to assess noninvasive optical coherence tomography (OCT) for early detection and evaluation of chemotherapy-induced oral mucositis in five patients. In five patients receiving neoadjuvant chemotherapy for primary breast cancer, oral mucositis was assessed clinically, and imaged using noninvasive OCT. Imaging was scored using a novel imaging-based scoring system. Conventional clinical assessment using the Oral Mucositis Assessment Scale was used as the gold standard. Patients were evaluated on days 0, 2, 4, 7, and 11 after commencement of chemotherapy. OCT images were visually examined by one blinded investigator. The following events were identified using OCT: (1) change in epithelial thickness and subepithelial tissue integrity (beginning on day 2), (2) loss of surface keratinized layer continuity (beginning on day 4), (3) loss of epithelial integrity (beginning on day 4). Imaging data gave higher scores compared to clinical scores earlier in treatment, suggesting that the imaging-based diagnostic scoring was more sensitive to early mucositic change than the clinical scoring system. Once mucositis was established, imaging and clinical scores converged. Chemotherapy-induced oral changes were identified prior to their clinical manifestation using OCT, and the proposed scoring system for oral mucositis was validated for the semiquantification of mucositic change.

ObjectiveBacterial factors, including strain type, anatomic distribution and density, and host responses are important determinants in the pathogenesis of erosive and neoplastic changes linked to gastric Helicobacter pylori (H. pylori) infection. The purpose of this study was to investigate the potential use of photodiagnostics in mapping H. pylori infection. The relationship between fluorescence in individual gastric pits of H. pylori(+) and H. pylori(-) subjects versus that in larger field views of the gastric mucosa and the use of fluorescence to determine H. pylori status in different gastric areas were studied.Material and methodsAntrum, corpus and fundus biopsies from 8 H. pylori(+) and 4 H. pylori(-) subjects taken during two gastroscopies were used for autofluorescence (535 nm excitation) and aminolevulinic acid (ALA)-induced protoporphyrin IX fluorescence (405 nm excitation) determinations.ResultsIn the antrum, corpus and fundus a close correlation between individual pit and full-field image (FFI) fluorescence was demonstrated for H. pylori status (R>0.85; R>0.75; R>0.80) and both excitation wavelengths (R>0.89; R>0.85; R>0.95), respectively. In the antrum, FFI in H. pylori(+) subjects exceeded that in H. pylori(-) subjects using 405 nm but not 535 nm excitation regardless of ALA treatment (pcorpus>antrum for both H. pylori(+) and H. pylori(-) subjects regardless of ALA treatment (p=0.03).ConclusionsFluorescence-based identification of areas of H. pylori-infected gastric mucosa using 405 nm excitation combined with ALA treatment is feasible and, using a ratio of 535 nm:405 nm-excited fluorescence, it is possible to distinguish H. pylori status and the different areas of the stomach even without ALA.

The Mueller matrix describes all the polarizing properties of a sample and, therefore, the optical differences between noncancerous and precancerous tissue that may be present within the matrix elements. A high-speed polarimetry system that generates 16 (4x4) full Mueller matrices to characterize tissues is presented. Feature extraction is done on the Mueller matrix elements resulting in depolarization and retardance images by polar decomposition. These are used to detect and classify early oral cancers and precancerous changes in epithelium such as dysplasia. These images are compared with orthogonal polarization images and analyzed in an attempt to identity useful factors for the differentiation between cancerous lesions and their benign counterparts. Our results indicate that polarimetry has potential as a method for the in vivo early detection and diagnosis of oral premalignancy.

PurposeTo assess noninvasive optical coherence tomography (OCT) and optical Doppler tomography (ODT) for early detection and evaluation of chemotherapy-induced oral mucositis.Experimental designCheek pouches of 10 Syrian golden hamsters were imaged using OCT/ODT during development of chemotherapy-induced mucositis. I.p. injections of 5-fluorouracil and mechanical irritation induced oral lesions. At 2, 4, 7, and 11 days, one hamster was sacrificed and processed for histopathology. OCT images were visually examined; ODT results were semiquantified. Imaging data were compared with histologic findings.ResultsDuring the development of mucositis, OCT/ODT identified the following events: (a) change in epithelial thickness (beginning on day 2), (b) loss of surface keratinized layer continuity (beginning on day 4), (c) loss of epithelial (day 4 onwards) and submucosal integrity (day 7 onwards), (d) changes in axial blood flow velocity (increased on days 2 and 4; decreased on day 7), and (e) changes in blood vessel size (diameter doubled on day 2; quadrupled on day 4; unchanged on day 7). The semiquantitative imaging-based scoring system identified the severity of mucositis as defined by histopathology. The combination of imaging criteria used allowed for the detection of early, intermediate, and late mucositic changes. Imaging data gave higher scores compared with clinical scores early on, suggesting that the imaging-based diagnostic scoring was more sensitive to early mucositic change than the clinical scoring system. Once mucositis was established, imaging and clinical scores converged.ConclusionOCT/ODT identified chemotherapy-induced oral changes before their clinical manifestation, and the proposed scoring system for oral mucositis was validated for the semiquantification of mucositic change.

ObjectiveTo assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage.MethodFresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination.ResultsNLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes.ConclusionThin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Background and objectivesThe degree of port wine stain (PWS) blanching following pulsed dye laser (PDL) therapy remains variable and unpredictable. Because of the limitations of current PDL therapy, alternative treatment approaches should be explored. The objective was to evaluate a novel methodology for selective vascular damage, combined photodynamic (PDT) and photothermal (PDL) treatment, using the in vivo chick chorioallantoic membrane (CAM) model.Study design/materials and methodsThirty microliters of benzoporphyrin derivative monoacid ring A (BPD) solution was administered intraperitoneally into chick embryos at day 12 of development. Study groups were: (1) control (no BPD, no light); (2) BPD alone; (3) continuous wave irradiation (CW) alone (576 nm, 60 mW/cm2, 125 seconds); (4) CW + PDL; (5) BPD+PDL; (6) PDT (BPD+CW); (7) PDL alone (585 nm, 4 J/cm(2)); and (8) PDT+PDL (BPD + CW followed immediately by PDL). Vessels were videotaped prior to, and at 1 hour post-intervention and then assessed for damage based on the following scale: 0, no damage; 1, coagulation; 1.5, vasoconstriction; 2.0, coagulation+vasoconstriction; 2.5, angiostasis; 3.0, hemorrhage. Damage scores were weighted by vessel "order."ResultsPDT + PDL resulted in significantly (P < 0.01) more severe vascular damage than was observed in any other study group: 127% more than PDT, 47% more than PDL alone.ConclusionsPDT + PDL is a novel and promising approach for selective vascular damage and may offer a more effective method for treatment of PWS and other vascular skin lesions.

The relative efficacy of Photofrin-based photodynamic therapy (PDT) has been compared with that of the second-generation photosensitizers 5-aminolevulinic acid (ALA), sulfonated chloro-aluminum phthalocyanine (AlPcSn), benzoporphyrin derivative monoacid ring A (BPD-MA), and lutetium texaphyrin (Lutex). PDT-induced vascular damage in the chick chorioallantoic membrane (CAM) is measured following topical application of the photosensitizers. In order to make meaningful comparisons, care is taken to keep treatment variables the same. These include light dose (5 and 10 J/cm2), power density (33 and 100 mW/cm2), and drug uptake time (30 and 90 min). The drug dose ranges from 0.1 microgram/cm2 for BPD to 5000 micrograms/cm2 for ALA. Results are also analyzed statistically according to CAM vessel type (arterioles versus venules), vessel diameter, and vessel development (embryonic age). For each photosensitizer, the order of importance for the various PDT parameters is found to be unique. The differences between the sensitizers are most likely due to variation in biophysical and biochemical characteristics, biodistribution, and uptake kinetics.

The aim of this study is to modify the chick chorioallantoic membrane (CAM) model into a whole-animal tumor model for photodynamic therapy (PDT). By using intraperitoneal (i.p.) photosensitizer injection of the chick embryo, use of the CAM for PDT has been extended to include systemic delivery as well as topical application of photosensitizers. The model has been tested for its capability to mimic an animal tumor model and to serve for PDT studies by measuring drug fluorescence and PDT-induced effects. Three second-generation photosensitizers have been tested for their ability to produce photodynamic response in the chick embryo/CAM system when delivered by i.p. injection: 5-aminolevulinic acid (ALA), benzoporphyrin derivative monoacid ring A (BPD-MA), and Lutetium-texaphyrin (Lu-Tex). Exposure of the CAM vasculature to the appropriate laser light results in light-dose-dependent vascular damage with all three compounds. Localization of ALA following i.p. injections in embryos, whose CAMs have been implanted with rat ovarian cancer cells to produce nodules, is determined in real time by fluorescence of the photoactive metabolite protoporphyrin IX (PpIX). Dose-dependent fluorescence in the normal CAM vasculature and the tumor implants confirms the uptake of ALA from the peritoneum, systemic circulation of the drug, and its conversion to PpIX.

Individual blood vessels in the chick chorioallantoic membrane (CAM) were selectively coagulated through photothermolysis, using pulsed laser irradiation at 585 nm. Pulse durations were chosen to be 0.45 ms and 10 ms, which correspond to the thermal relaxation times in blood vessels of 30 μm and 150 μm diameter, respectively. The short pulses, at an energy density (or light dose) D=3 Jcm-2, caused permanent occlusion of vessels of 40 μm diameter or less, while larger caliber vessels (60-120 p.m) required D=4-5 Jcm-2. The long-duration pulses, at D=7 Jcm-2, caused coagulation of the larger diameter vessels; the small caliber vessels and capillaries showed resistance to photothermolysis and required multiple exposures to achieve coagulation. The dose vs diameter (D vs d) relationship for coagulation was calculated for the two pulse shapes. The energy deposited in a cylindrical absorber of diameter d by an optical field, incident perpendicular to the vessel, was expressed analytically and compared with the energy required to coagulate a blood vessel of the same lumen diameter. When thermal diffusion is incorporated into the model, our findings can be accounted for quantitatively. This information will be of use for improving the laser treatment of port wine stains and other vasculopathies. A surprising observation was that arterioles were damaged at lower incident energy densities than venules having the same lumen diameter, despite the fact that absorbance in oxygen-rich and oxygen-poor blood is the same for 585 nm radiation.