Detecting head and neck lymph node metastases with white light reflectance spectroscopy; a pilot study

Introduction: A challenge in the treatment of patients with head and neck cancer is the management of occult cervical lymph node (LN) metastases. Single-fiber reflectance (SFR) spectroscopy has the potential to detect physiological tissue changes that occur in a positive LN. This pilot study aimed to investigate whether SFR spectroscopy could serve as an alternative or additional technique to detect cervical lymph node metastases. Materials and Methods: We performed intraoperative SFR spectroscopy measurements of LNs with and without malignancies. We analyzed if physiological and scattering parameters were significantly altered in positive LNs. Results: Nine patients with a total of nineteen LNs were included. Three parameters, blood volume fraction (BVF), microvascular saturation (StO2), and Rayleigh amplitude, were significantly lower in positive LNs. They were combined into one optical parameter ‘delta’, using discriminant analysis. Delta was significantly decreased in positive LNs, p = 0,0006. It had a high diagnostic accuracy where the sensitivity, specificity, PPV, and NPV were 90,0%, 88.9%, 90,0%, and 88.9%, respectively. The area under the ROC curve was 96.7% (95% confidence interval 89.7–100.0%). Conclusion: This proof of principle study is a first step in the development of an SFR spectroscopy technique to detect LN metastases in real time. A next step towards this goal is replicating these results in LNs with smaller metastases and in a larger cohort of patients. This future study will combine SFR spectroscopy with fine-needle aspiration, using the same needle, to perform preoperative in vivo measurements.</p

In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra (hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dualwavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed

Increase in protoporphyrin IX after 5-aminolevulinic acid based photodynamic therapy is due to local re-synthesis

Protoporphyrin IX (PpIX) fluorescence that is bleached during aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) increases again in time after treatment. In the present study we investigated if this increase in PpIX fluorescence after illumination is the result of local re-synthesis or of systemic redistribution of PpIX. We studied the spatial distribution of PpIX after PDT with and without cooling using the skin-fold observation chamber model. We were unable to show a correlation between the local PpIX fluorescence increase and the distance from a blood vessel. The spatial distribution of PpIX fluorescence within normal tissue or tumour is not changed in response to the illumination. These observations suggest that there is no diffusion of PpIX into the treated tissue. Cooling the tissue to 12 degrees C, a temperature at which PpIX synthesis is inhibited, inhibited the PpIX fluorescence increase normally observed after illumination. We also found a strong correlation between local PpIX photobleaching during illumination and the fluorescence intensity 1 h after illumination similar to what we have observed in patients treated with ALA-PDT. Therefore we conclude that the increase in PpIX fluorescence after illumination is due to local cellular re-synthesi

Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.5, 24 and 48 h after PDT. As expected high fluorescence intensities were observed in the epidermis and pilosebaceous units and no fluorescence in the cutaneous musculature after both MAL and ALA application. The dermis showed localised fluorescence that corresponds to the cytoplasma of dermal cells like fibroblast and mash cells. Spectral analysis showed a typical PpIX fluorescence spectrum confirming that it is PpIX fluorescence. There was no clear difference in the depth and spatial distribution of PpIX fluorescence between the two precursors in these normal mouse skin samples. This result combined with the conclusion of Moan et al. that ALA but not MAL is systemically distributed after topical application on mouse skin [Moan et al., Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters, Int. J. Cancer 103 (2003) 132-135] suggests that endothelial cells are involved in increased response of tissues to ALA-PDT using light fractionation. Histological analysis 2.5 h after PDT showed more edema formation after ALA-PDT compared to MAL-PDT that was not accompanied by a difference in the inflammatory response. This suggests that endothelial cells respond differently to ALA and MAL-PDT. Further investigation is needed to determine the role of endothelial cells in ALA-PDT and the underlying mechanism behind the increased effectiveness of light fractionation using a dark interval of 2 h found after ALA but not after MAL-PDT. (c) 2008 Elsevier B.V. All rights reserve

Evidence for a bystander role of neutrophils in the response to systemic 5-aminolevulinic acid-based photodynamic therapy

A significant increase in the number of circulating and tumour neutrophils immediately after therapy was observed while investigating the increase in response of tissues to aminolevulinic acid-based photodynamic therapy (ALA-PDT) using a twofold illumination scheme with a prolonged dark interval. The action of (tumour) neutrophils is an important therapeutic adjunct to the deposition of singlet oxygen within the treatment volume, for many photosensitizers. It is not known if those phagocytes contribute to the improved outcome of ALA-PDT. In this study we investigated the role of neutrophils in the response to PDT using systemic ALA with and without light fractionation. Rhabdomyosarcoma, transplanted in the thigh of female WAG/Rij rats were illuminated transdermally using 633 nm light following i.v. administration of 200 mg/kg ALA. The pharmacokinetics of protoporphyrin IX (PpIX) within the tumour tissue during therapy were determined to compare with that observed in other models for topical administration of ALA. PDT was performed under immunologically normal or neutropenic conditions using various illumination schemes. The number of neutrophils in tumour and in the circulation were determined as a function of time after treatment and compared with growth delay of each scheme. Fluorescence spectroscopy revealed similar pharmacokinetics of PpIX to those observed during and after topical ALA-PDT. The number of neutrophils within the illuminated tumour and in the circulation increased significantly following therapy. This increase in the number of neutrophils was associated with an increase in the efficacy of therapy: the more effective the therapy the greater the increase in tumour and blood neutrophils. Administration of anti-granulocyte serum treatment prevented the influx of neutrophils after ALA-PDT, but did not lead to a significant decrease in the efficacy of the PDT treatment on the growth of the tumour for any illumination scheme investigated. These results indicate that the magnitude of damage inflicted on the tumour by ALA-PDT does not depend on the presence of neutrophils in the tumour or circulation and that the role of neutrophils in ALA-PDT is much less important than in PDT using other photosensitizers. These data contribute to the understanding of the mechanism of response of tissue to systemic ALA-PD

Did ASEAN function in its early years? : A focus on the institutional structures that constrained Indonesia's expanding influence in the region, 1966-1968

BACKGROUND:Light fractionation significantly increases the efficacy of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) using the nano-emulsion based gel formulation BF-200. PDT using BF-200 ALA has recently been clinically approved and is under investigation in several phase III trials for the treatment of actinic keratosis. This study is the first to compare BF-200 ALA with ALA in preclinical models. RESULTS:In hairless mouse skin there is no difference in the temporal and spatial distribution of protoporphyrin IX determined by superficial imaging and fluorescence microscopy in frozen sections. In the skin-fold chamber model, BF-200 ALA leads to more PpIX fluorescence at depth in the skin compared to ALA suggesting an enhanced penetration of BF-200 ALA. Light fractionated PDT after BF-200 ALA application results in significantly more visual skin damage following PDT compared to a single illumination. Both ALA formulations show the same visual skin damage, rate of photobleaching and change in vascular volume immediately after PDT. Fluorescence immunohistochemical imaging shows loss of VE-cadherin in the vasculature at day 1 post PDT which is greater after BF-200 ALA compared to ALA and more profound after light fractionation compared to a single illumination. DISCUSSION:The present study illustrates the clinical potential of light fractionated PDT using BF-200 ALA for enhancing PDT efficacy in (pre-) malignant skin conditions such as basal cell carcinoma and vulval intraepithelial neoplasia and its application in other lesion such as cervical intraepithelial neoplasia and oral squamous cell carcinoma where current approaches have limited efficacy

Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope

Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and noncentrosymmetric-structured proteins is rapidly gaining interest in biomedical applications. The advantage of this technique includes high imaging penetration depth and minimal phototoxic effects on tissues. Because fluorescent dyes are not used, discrimination between different components within the tissue is challenging. We have developed a nonlinear spectral imaging microscope based on a home-built multiphoton microscope, a prism spectrograph, and a high-sensitivity CCD camera for detection. The sensitivity of the microscope was optimized for autofluorescence and second harmonic imaging over a broad wavelength range. Importantly, the spectrograph lacks an entrance aperture; this improves the detection efficiency at deeper lying layers in the specimen. Application to the imaging of ex vivo and in vivo mouse skin tissues showed clear differences in spectral emission between skin tissue layers as well as biochemically different tissue components. Acceptable spectral images could be recorded up to an imaging depth of similar to 100 mu m. (C) 2008 Society of Photo-Optical Instrumentation Engineer

Ex vivo quantification of mTHPC concentration in tissue: Influence of chemical extraction on the optical properties

A method for the quantification of the concentration of the photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) in tissue samples is presented. The technique is an extension of a previously published method based on alkaline hydrolysis of tissue, using Solvable (TM) as a tissue solubilizer. mTHPC quantification was achieved by subsequent fluorescence spectroscopy. Since the original extraction method involved multiple steps in which water dilution of the sample was implemented, we studied the spectral characteristics of mTHPC in different Solvable (TM)/water mixtures. Using UV-VIS absorption and fluorescence spectroscopy, it was demonstrated that the spectral characteristics of mTHPC vary for different Solvable (TM) concentrations. In the range of 20-100% Solvable (TM), the fluorescence intensity of mTHPC did not change, while dramatic changes in the mTHPC fluorescence intensity were observed for lower Solvable (TM) concentrations ( <20%) due to increasing hydrophilicity of the environment, combined with pH alterations. We also demonstrated that the absorption and fluorescence spectra of the dissolved tissue were time-dependent. Longer incubation of the samples resulted in a significant increase of the native tissue chromophore fluorescence. This implies that for the correct quantification of photosensitizer concentrations, the fluorescence of native tissue chromophores must be accounted for. (C) 2008 Elsevier B.V. All rights reserve