Intradermal Indocyanine Green for In Vivo Fluorescence Laser Scanning Microscopy of Human Skin: A Pilot Study

BACKGROUND: In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. METHODOLOGY/PRINCIPAL FINDINGS: Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG) is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. CONCLUSIONS/SIGNIFICANCE: Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of ICG in combination with the near infrared laser opens new ways for minimal-invasive diagnosis and monitoring of skin disorders

Automatic Quantification of Epidermis Curvature in H&E Stained Microscopic Skin Image of Mice

Changes in the curvature of the epidermis layer is often associated with many skin disorders, such as ichthyoses and generic effects of ageing. Therefore, methods to quantify changes in the curvature are of a scientific and clinical interest. Manual methods to determine curvature are both laborious and intractable to large scale investigations. This paper proposes an automatic algorithm to quantify curvature of microscope images of H&E-stained murine skin. The algorithm can be divided into three key stages. First, skin layers segmentation based on colour deconvolution to separate the original image into three channels of different representations to facilitate segmenting the image into multiple layers, namely epidermis, dermis and subcutaneous layers. The algorithm then further segments the epidermis layer into cornified and basal sub-layers. Secondly, it quantifies the curvature of the epidermis layer by measuring the difference between the epidermis edge and a straight line (theoretical reference line) connecting the two far sides of the epidermis edge. Finally, the curvature measurements extracted from a large number of images of mutant mice are used to identify a list of genes responsible for changes in the epidermis curvature. A dataset of 5714 H&E microscopic images of mutant and wild type mice were used to evaluate the effectiveness of the algorithm

Universal in vivo Textural Model for Human Skin based on Optical Coherence Tomograms

Currently, diagnosis of skin diseases is based primarily on visual pattern recognition skills and expertise of the physician observing the lesion. Even though dermatologists are trained to recognize patterns of morphology, it is still a subjective visual assessment. Tools for automated pattern recognition can provide objective information to support clinical decision-making. Noninvasive skin imaging techniques provide complementary information to the clinician. In recent years, optical coherence tomography has become a powerful skin imaging technique. According to specific functional needs, skin architecture varies across different parts of the body, as do the textural characteristics in OCT images. There is, therefore, a critical need to systematically analyze OCT images from different body sites, to identify their significant qualitative and quantitative differences. Sixty-three optical and textural features extracted from OCT images of healthy and diseased skin are analyzed and in conjunction with decision-theoretic approaches used to create computational models of the diseases. We demonstrate that these models provide objective information to the clinician to assist in the diagnosis of abnormalities of cutaneous microstructure, and hence, aid in the determination of treatment. Specifically, we demonstrate the performance of this methodology on differentiating basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) from healthy tissue

Automated Biochemical, Morphological, and Organizational Assessment of Precancerous Changes from Endogenous Two-Photon Fluorescence Images

Multi-photon fluorescence microscopy techniques allow for non-invasive interrogation of live samples in their native environment. These methods are particularly appealing for identifying pre-cancers because they are sensitive to the early changes that occur on the microscopic scale and can provide additional information not available using conventional screening techniques.In this study, we developed novel automated approaches, which can be employed for the real-time analysis of two-photon fluorescence images, to non-invasively discriminate between normal and pre-cancerous/HPV-immortalized engineered tissues by concurrently assessing metabolic activity, morphology, organization, and keratin localization. Specifically, we found that the metabolic activity was significantly enhanced and more uniform throughout the depths of the HPV-immortalized epithelia, based on our extraction of the NADH and FAD fluorescence contributions. Furthermore, we were able to separate the keratin contribution from metabolic enzymes to improve the redox estimates and to use the keratin localization as a means to discriminate between tissue types. To assess morphology and organization, Fourier-based, power spectral density (PSD) approaches were employed. The nuclear size distribution throughout the epithelial depths was quantified by evaluating the variance of the corresponding spatial frequencies, which was found to be greater in the normal tissue compared to the HPV-immortalized tissues. The PSD was also used to calculate the Hurst parameter to identify the level of organization in the tissues, assuming a fractal model for the fluorescence intensity fluctuations within a field. We found the range of organization was greater in the normal tissue and closely related to the level of differentiation.A wealth of complementary morphological, biochemical and organizational tissue parameters can be extracted from high resolution images that are acquired based entirely on endogenous sources of contrast. They are promising diagnostic parameters for the non-invasive identification of early cancerous changes and could improve significantly diagnosis and treatment for numerous patients

The Advanced Applications For Optical Coherence Tomography In Skin Imaging

Optical coherence tomography (OCT), based on the principle of interferometry, is a fast and non-invasive imaging modality, which has been approved by FDA for dermatologic applications. OCT has high spatial resolution up to micrometer scale compared to traditional ultrasound imaging. In addition, OCT can provide real-time cross-sectional images with 1 to 2 mm penetration depth, which makes it an ideal imaging technique to assess the skin micro-morphology and pathology without any tissue removal. Many studies have investigated the possibilities of using OCT to evaluate dermatologic conditions, such as skin cancer, dermatitis, psoriasis, and skin damages. Hence, OCT has tremendous potential to provide skin histological and pathological information and assist differential diagnosis of various skin diseases. In this study, we used a swept-source OCT with 1305 nm central wavelength to explore its advanced applications in dermatology. This dissertation consists of four major research projects. First, we explored the feasibility of OCT imaging for assisting real-time visualization in skin biopsy. We showed that OCT could be used to guide and track a needle insertion in mouse skin in real-time. The structure of skin and the movement of needle can be clearly seen on the OCT images without any time delay during the procedures. Next, we tested the concept of performing the punch biopsy using OCT hand-held probe attached to a piercing tip in a phantom. We proved that using the OCT is a reliable technique to delineate the margin of lesion in phantom. And it is possible to perform the punch biopsy with the OCT probe. Second, we tested the performance of contrast-enhanced OCT in melanoma detection in an in vitro study. Melanoma is the most lethal type of skin cancer. Early detection could significantly improve the long-term survival rate of patients. In this initial study, a contrast agent (Gal3-USGNPs) is developed by conjugating melanoma biomarker (Gal3) to ultra-small gold nanoparticles (USGNPs). We showed that the contrast agent can differentiate B16 melanoma cells from normal skin keratinocytes in vitro. To avoid systemic administration of USGNPs, the third project continues to explore the enhanced topical delivery of USGNPs. In this study, we used OCT to monitor the topical delivery of nanoparticles on pig skin over time. And the diffusion and penetration of USGNPs in skin can be improved by applying chemical and physical enhancers such as DMSO and sonophoresis. Finally, in addition to image the cross-sectional structure of skin, we also aim to extract quantitative information from OCT images. The skin optical properties such as attenuation coefficient can be measured from OCT images. We measured and compared the skin attenuation coefficient in the skin of forehead and lateral hip, the skin of three different age groups, and the skin of three different Fitzpatrick types. The statistical analysis showed that epidermis has much higher attenuation coefficient than dermis. And the skin type V & VI have a relatively lower attenuation coefficient than the other skin types. These studies could aid the detection of skin cancer using imaging techniques and provide some new insights into the future applications of OCT in dermatology

Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

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Melanoma and nevi subtype histopathological characterization with optical coherence tomography

Background: Melanoma incidence has continued to rise in the latest decades, and the forecast is not optimistic. Non-invasive diagnostic imaging techniques such as optical coherence tomography (OCT) are largely studied; however, there is still no agreement on its use for the diagnosis of melanoma. For dermatologists, the differentiation of non-invasive (junctional nevus, compound nevus, intradermal nevus, and melanoma in-situ) versus invasive (superficial spreading melanoma and nodular melanoma) lesions is the key issue in their daily routine. Methods: This work performs a comparative analysis of OCT images using haematoxylin-eosin (HE) and anatomopathological features identified by a pathologist. Then, optical and textural properties are extracted from OCT images with the aim to identify subtle features that could potentially maximize the usefulness of the imaging technique in the identification of the lesion?s potential invasiveness. Results: Preliminary features reveal differences discriminating melanoma in-situ from superficial spreading melanoma and also between melanoma and nevus subtypes that pose a promising baseline for further research. Conclusions: Answering the final goal of diagnosing non-invasive versus invasive lesions with OCT does not seem feasible in the short term, but the obtained results demonstrate a step forward to achieve this.This work has been funded by the Department of Economic Development, Sustainability and the Environment of the Basque Government (Spain) ELKARTEK projects ONKOTOOLS with grant numbers KK-2020/00069, the Spanish Ministry of Science and Education CERVERA project AI4ES with grant numbers CER-20211030, and by the ECSEL JU European project ASTONISH with the grant number 692470, UC Industrial Doctorate DI14