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Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique in which A-scans are acquired in parallel through line illumination with a broadband laser and line detection with a line-scan camera. B-scan imaging at high spatial resolution is achieved by dynamic focusing in a Linnik interferometer. This paper presents an LC-OCT device based on a custom-designed Mirau interferometer that offers similar spatial resolution and detection sensitivity. The device has the advantage of being more compact and lighter. In vivo imaging of human skin with a resolution of 1.3 µm × 1.1 µm (lateral × axial) is demonstrated over a field of 0.9 mm × 0.4 mm (lateral × axial) at 12 frames per second.
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BACKGROUND: Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique providing "optical biopsies" of the skin in real time and non-invasively. At a center optical wavelength of 1.3 µm, this innovative technology can be applied to dermo-cosmetic product development due to both high image resolution (~2 µm) and sufficient penetration (~0.5 mm). Nevertheless, the precise dermal area analyzed with LC-OCT has never been identified. In this study, the objective was to compare LC-OCT images with histological sections of the same area, in order to validate a new method for in vivo and non-invasive quantification of superficial dermis thickness. Once validated, this standardized and quantitative method was used to assess age-related changes of the superficial dermis. MATERIALS AND METHODS: Ex vivo LC-OCT acquisitions and hematoxylin-eosin-safran staining were performed on a panel of four healthy Caucasian female volunteers. In vivo LC-OCT study of skin aging was performed on a panel of 37 healthy Caucasian female divided into five different age-groups. RESULTS: Comparison with histological sections revealed that LC-OCT images allow the visualization and the quantification of the superficial portion of papillary dermis. Applied to different age-group of volunteers, LC-OCT images show a constant decrease in this superficial dermis thickness with age. CONCLUSIONS: In conclusion, we have introduced LC-OCT as a novel technique for in vivo and non-invasive evaluation of superficial dermis thickness. This approach could be used in the future to demonstrate visually and quantitatively the capacity of a dermo-cosmetic active ingredient to renormalize the structural properties of the dermis.
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Derme/diagnóstico por imagem , Derme/patologia , Técnicas Histológicas/normas , Tomografia de Coerência Óptica/métodos , Adulto , Idoso , Biópsia/instrumentação , Cosméticos , Feminino , Técnicas Histológicas/estatística & dados numéricos , Humanos , Pessoa de Meia-Idade , Envelhecimento da Pele/patologia , Tomografia de Coerência Óptica/estatística & dados numéricosRESUMO
A time-domain optical coherence tomography technique is introduced for high-resolution B-scan imaging in real-time. The technique is based on a two-beam interference microscope with line illumination and line detection using a broadband spatially coherent light source and a line-scan camera. Multiple (2048) A-scans are acquired in parallel by scanning the sample depth while adjusting the focus. Quasi-isotropic spatial resolution of 1.3 µm × 1.1 µm (lateral × axial) is achieved. In vivo cellular-level resolution imaging of human skin is demonstrated at 10 frames per second with a penetration depth of â¼500 µm.
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Full-field optical coherence microscopy (FFOCM) is an optical technique, based on low-coherence interference microscopy, for tomographic imaging of semi-transparent samples with micrometer-scale spatial resolution. The differences in refractive index between the sample and the immersion medium of the microscope objectives may degrade the FFOCM image quality because of focus defect and optical dispersion mismatch. These phenomena and their consequences are discussed in this theoretical paper. Experimental methods that have been implemented in FFOCM to minimize the adverse effects of these phenomena are summarized and compared.
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High-resolution full-field optical coherence microscopy (FF-OCM) is demonstrated using a single broadband light-emitting diode (LED). The characteristics of the LED-illumination FF-OCM system are measured and compared to those obtained using a halogen lamp, the light source of reference in FF-OCM. Both light sources yield identical performance in terms of spatial resolution and detection sensitivity, using the same setup and camera. In particular, an axial resolution of 0.7 µm (in water) is reached. A Xenopus laevis tadpole and ex-vivo human skin have been imaged using both sources, resulting in similar images, showing for the first time that LEDs could favorably replace halogen lamps in high-resolution FF-OCM for biomedical imaging.
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Full-field optical coherence microscopy (FF-OCM) with isotropic spatial resolution of 0.5 µm (in water), at 700 nm center wavelength, is reported. A theoretical study of the FF-OCM axial response is carried out for maximizing the axial resolution of the system, considering the effect of optical dispersion. The lateral resolution is optimized by using water-immersion microscope objectives with a numerical aperture of 1.2. This ultrahigh-resolution FF-OCM system is applied to animal and human skin tissue imaging, revealing ultra-fine in-depth structures at the sub-cellular level.
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An original single-objective, full-field optical coherence microscopy system is reported that is capable of imaging both the phase and the amplitude of semi-transparent samples over a field of view of 17.5 mm×17.5 mm with an axial sectioning resolution of 1.5 µm. A special stack acquisition arrangement ensures optimal reachable imaging depth. Several phase-shifting interferometry algorithms for phase measurement with broadband light are compared theoretically and experimentally. Using the phase information, noninvasive depth-resolved topographic images of multilayer samples are produced to characterize each layer by measuring their defects and curvature with a nanometric scale precision. Using the amplitude information, tomographic images with a constant detection sensitivity of â¼80 dB through the entire field of view are obtained and applied to biological specimens.
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Full-field optical coherence microscopy is an established optical technology based on low-coherence interference microscopy for high-resolution imaging of semitransparent samples. In this Letter, we demonstrate an extension of the technique using a visible to short-wavelength infrared camera and a halogen lamp to image in three distinct bands centered at 635, 870, and 1170 nm. Reflective microscope objectives are employed to minimize chromatic aberrations of the imaging system operating over a spectral range extending from 530 to 1700 nm. Constant 1.9-µm axial resolution (measured in air) is achieved in each of the three bands. A dynamic dispersion compensation system is set up to preserve the axial resolution when the imaging depth is varied. The images can be analyzed in the conventional RGB color channels representation to generate three-dimensional images with enhanced contrast. The capability of the system is illustrated by imaging different samples.
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We propose a 3D imaging technique based on the combination of full-field swept-source optical coherence microscopy (FF-SSOCM) with low spatial coherence illumination and a special numerical processing that allows for numerically focused coherent-noise-free imaging without mechanical scanning in longitudinal or transversal directions. We show, both theoretically and experimentally, that the blurring effects arising in FF-SSOCM due to defocus can be corrected by appropriate numerical processing even when low spatial coherence illumination is used. A FF-SSOCM system was built for testing the performance of this technique. Coherent-noise-free imaging of a sample with longitudinal extent exceeding the optical depth of field is demonstrated without displacement of the sample or any optical element.
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Line-field confocal optical coherence tomography (LC-OCT) is a non-invasive optical imaging technique based on a combination of the optical principles of optical coherence tomography and reflectance confocal microscopy with line-field illumination, which can generate cell-resolved images of the skin, in vivo, in vertical section, horizontal section and in three dimensions. This article reviews the optical principles of LC-OCT, including low coherence interferometry, confocal filtering and line-field arrangement. The optical setup allowing for the acquisition of color images of the skin surface in parallel with LC-OCT images, without compromising LC-OCT performance, is also presented. Practical use of LC-OCT is demonstrated through an overview of the workflow of examining a patient using a commercial handheld LC-OCT probe (deepLive™, DAMAE Medical), from creating the patient record in the software, acquiring the images, to reviewing and interpreting the images. LC-OCT can generate a significant amount of data, making automated deep learning algorithms particularly relevant for assisting in the analysis of LC-OCT images. A review of algorithms developed for skin layer segmentation, keratinocyte nuclei segmentation, and automatic detection of atypical keratinocyte nuclei is provided.
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Interpretação de Imagem Assistida por Computador , Pele , Tomografia de Coerência Óptica , Tomografia de Coerência Óptica/métodos , Pele/diagnóstico por imagem , Humanos , Microscopia Confocal , Algoritmos , QueratinócitosRESUMO
Line-field confocal optical coherence tomography (LC-OCT) is a non-invasive optical imaging technique based on a combination of the principles of optical coherence tomography and reflectance confocal microscopy with line-field illumination, which can generate cell-resolved images of the skin in vivo. This article reports on the LC-OCT technique and its application in dermatology. The principle of the technique is described, and the latest technological innovations are presented. The technology has been miniaturized to fit within an ergonomic handheld probe, allowing for the easy access of any skin area on the body. The performance of the LC-OCT device in terms of resolution, field of view, and acquisition speed is reported. The use of LC-OCT in dermatology for the non-invasive detection, characterization, and therapeutic follow-up of various skin pathologies is discussed. Benign and malignant melanocytic lesions, non-melanocytic skin tumors, such as basal cell carcinoma, squamous cell carcinoma and actinic keratosis, and inflammatory and infectious skin conditions are considered. Dedicated deep learning algorithms have been developed for assisting in the analysis of LC-OCT images of skin lesions.
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Full-field optical coherence tomography (FF-OCT) is a recent optical imaging technology based on low-coherence interference microscopy for imaging of semi-transparent samples with ~1 µm spatial resolution. FF-OCT produces en-face tomographic images obtained by arithmetic combination of interferometric images acquired by an array camera. In this paper, we demonstrate a unique multimodal FF-OCT system, capable of measuring simultaneously the intensity, the power spectrum and the phase-retardation of light backscattered by the sample being imaged. Compared to conventional FF-OCT, this multimodal system provides enhanced imaging contrasts at the price of a moderate increase in experimental complexity and cost.
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Aumento da Imagem/instrumentação , Microscopia de Interferência/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Análise Espectral/instrumentação , Tomografia de Coerência Óptica/instrumentação , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy. Complementary image data are provided by the dual modality instrument in the context of biological tissue screening. FF-OCM imaging modality shows the tissue microarchitecture, while fluorescence microscopy highlights specific tissue features with cellular-level resolution by using targeting contrast agents. Complementary tissue morphology and biochemical features could potentially improve the understanding of cellular functions and disease diagnosis.
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Iluminação/métodos , Microscopia de Fluorescência/métodos , Fenômenos Ópticos , Tomografia de Coerência Óptica/métodos , Animais , Colo/citologia , Camundongos , Fatores de TempoRESUMO
Line-field confocal optical coherence tomography (LC-OCT) is an optical modality that provides three-dimensional (3D) images of the skin at cellular resolution. Confocal Raman microspectroscopy (CRM) is a label-free optical technique that can provide point measurement of the molecular content of the skin. This work presents a method to co-localize LC-OCT and CRM acquisitions for morpho-molecular analysis of ex vivo skin tissues at cellular level. The co-localization method allows acquisition of Raman spectra at specific locations in a sample identified from a 3D LC-OCT image, with an accuracy of ± 20 µm. The method was applied to the characterization of tattooed skin biopsies with adverse tattoo reactions. LC-OCT images allowed to target specific regions in the biopsies where the presence of tattoo ink was revealed by detection of the Raman signature of ink pigments. Micrometer-sized foreign bodies of various materials as well as inflammatory cells were also identified within the biopsies. From these results, we demonstrate the value of the LC-OCT-CRM co-localization method and its potential for future ex vivo analysis of suspicious skin lesions.
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SIGNIFICANCE: Line-field confocal optical coherence tomography (LC-OCT) is a recently introduced high-resolution imaging modality based on a combination of low-coherence optical interferometry and reflectance confocal optical microscopy with line illumination and line detection. Capable of producing three-dimensional (3D) images of the skin with cellular resolution, in vivo, LC-OCT has been mainly applied in dermatology and dermo-cosmetology. The LC-OCT devices capable of acquiring 3D images reported so far are based on a Linnik interferometer using two identical microscope objectives. In this configuration, LC-OCT cannot be designed to be a very compact and light device, and the image acquisition speed is limited. AIM: The objective of this work was to develop a more compact and lighter LC-OCT device that is capable of acquiring images faster without significant degradation of the resolution and with optimized detection sensitivity. APPROACH: We developed an LC-OCT device based on a Mirau interferometer using a single objective. Dynamic adjustment of the camera frequency during the depth scan is implemented, using a faster camera and a more powerful light source. The reflectivity of the beam-splitter in the Mirau interferometer was optimized to maximize the detection sensitivity. A galvanometer scanner was incorporated into the device for scanning the illumination line laterally. A stack of adjacent B-scans, constituting a 3D image, can thus be acquired. RESULTS: The device is able to acquire and display B-scans at 17 fps. 3D images with a quasi-isotropic resolution of â¼1.5 µm (1.3, 1.9, and 1.1 µm in the x , y, and z directions, respectively) over a field of 940 µm × 600 µm × 350 µm (x × y × z) can be obtained. 3D imaging of human skin at cellular resolution, in vivo, is reported. CONCLUSIONS: The acquisition rate of the B-scans, at 17 fps, is unprecedented in LC-OCT. Compared with the conventional LC-OCT devices based on a Linnik interferometer, the reported Mirau-based LC-OCT device can acquire B-scans â¼2 times faster. With potential advantages in terms of compactness and weight, a Mirau-based device could easily be integrated into a smaller and lighter handheld probe for use by dermatologists in their daily medical practice.
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Interferometria , Tomografia de Coerência Óptica , Humanos , Imageamento Tridimensional/métodos , Microscopia Confocal , Pele/diagnóstico por imagem , Tomografia de Coerência Óptica/métodosRESUMO
Diagnosis based on histopathology for skin cancer detection is today's gold standard and relies on the presence or absence of biomarkers and cellular atypia. However it suffers drawbacks: it requires a strong expertise and is time-consuming. Moreover the notion of atypia or dysplasia of the visible cells used for diagnosis is very subjective, with poor inter-rater agreement reported in the literature. Lastly, histology requires a biopsy which is an invasive procedure and only captures a small sample of the lesion, which is insufficient in the context of large fields of cancerization. Here we demonstrate that the notion of cellular atypia can be objectively defined and quantified with a non-invasive in-vivo approach in three dimensions (3D). A Deep Learning (DL) algorithm is trained to segment keratinocyte (KC) nuclei from Line-field Confocal Optical Coherence Tomography (LC-OCT) 3D images. Based on these segmentations, a series of quantitative, reproducible and biologically relevant metrics is derived to describe KC nuclei individually. We show that, using those metrics, simple and more complex definitions of atypia can be derived to discriminate between healthy and pathological skins, achieving Area Under the ROC Curve (AUC) scores superior than 0.965, largely outperforming medical experts on the same task with an AUC of 0.766. All together, our approach and findings open the door to a precise quantitative monitoring of skin lesions and treatments, offering a promising non-invasive tool for clinical studies to demonstrate the effects of a treatment and for clinicians to assess the severity of a lesion and follow the evolution of pre-cancerous lesions over time.
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Aprendizado Profundo , Patologia/métodos , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Feminino , Técnicas Histológicas , Humanos , Imageamento Tridimensional , Queratinócitos/química , Queratinócitos/patologia , Masculino , Pessoa de Meia-Idade , Patologia/instrumentação , Pele/diagnóstico por imagem , Pele/patologia , Neoplasias Cutâneas/diagnóstico , Tomografia de Coerência Óptica/métodosRESUMO
Epidermal three-dimensional (3D) topography/quantification has not been completely characterized yet. The recently developed line-field confocal optical coherence tomography (LC-OCT) provides real-time, high-resolution, in-vivo 3D imaging of the skin. This pilot study aimed at quantifying epidermal metrics (epidermal thicknesses, dermal-epidermal junction [DEJ] undulation and keratinocyte number/shape/size) using 3D LC-OCT. For each study participant (8 female, skin-type-II, younger/older volunteers), seven body sites were imaged with LC-OCT. Epidermal metrics were calculated by segmentations and measurements assisted by artificial intelligence (AI) when appropriate. Thicknesses of epidermis/SC, DEJ undulation and keratinocyte nuclei volume varied across body sites. Evidence of keratinocyte maturation was observed in vivo: keratinocyte nuclei being small/spherical near the DEJ and flatter/elliptical near the skin surface. Skin microanatomy can be quantified by combining LC-OCT and AI. This technology could be highly relevant to understand aging processes and conditions linked to epidermal disorders. Future clinical/research applications are to be expected in this scenario.
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Inteligência Artificial , Tomografia de Coerência Óptica , Epiderme/diagnóstico por imagem , Feminino , Humanos , Projetos Piloto , Pele , Tomografia de Coerência Óptica/métodosRESUMO
Significant motion artifacts limit the performance of conventional full-field optical coherence tomography (FF-OCT) for in-vivo imaging. We present a theoretical and experimental study of those limitations. A new FF-OCT system suppressing most of artifacts due to sample motions is demonstrated using instantaneous phase shifting with nonpolarizing optics and pulsed illumination. The experimental setup is based on a Linnik-type interferometer illuminated by the superluminescence emission from a Ti:Al(2)O(3) waveguide crystal. En face tomographic images are calculated as a combination of two phase-opposed interferometric images acquired simultaneously by two CCD cameras placed at both outputs of the interferometer, with a spatial resolution of 0.8 microm x 1.6 microm (axial x transverse) and a detection sensitivity of approximately 60 dB.
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Tomografia de Coerência Óptica/métodos , Artefatos , Imageamento Tridimensional , Interferometria , Modelos Teóricos , Movimento (Física) , Dispositivos Ópticos , Fenômenos Ópticos , Tomografia de Coerência Óptica/instrumentação , Tomografia de Coerência Óptica/estatística & dados numéricosRESUMO
Line-field confocal optical coherence tomography (LC-OCT) is a recently introduced technique for ultrahigh-resolution vertical section (B-scan) imaging of human skin in vivo. This work presents a new implementation of the LC-OCT technique to obtain horizontal section images (C-scans) in addition to B-scans. C-scan imaging is achieved with this dual-mode LC-OCT system using a mirror galvanometer for lateral scanning along with a piezoelectric chip for modulation of the interferometric signal. A quasi-identical spatial resolution of â¼ 1 µm is measured for both B-scans and C-scans. The images are acquired in both modes at a rate of 10 frames per second. The horizontal field of view of the C-scans is 1.2 × 0.5 mm2, identical to the vertical field of view of the B-scans. The user can switch between the two modes by clicking a button. In vivo cellular-resolution imaging of human skin is demonstrated in both B-scan and C-scan modes, with the possibility to navigate within the skin tissues in real time.
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This paper reports on the latest advances in line-field confocal optical coherence tomography (LC-OCT), a recently invented imaging technology that now allows the generation of either horizontal (x × y) section images at an adjustable depth or vertical (x × z) section images at an adjustable lateral position, as well as three-dimensional images. For both two-dimensional imaging modes, images are acquired in real-time, with real-time control of the depth and lateral positions. Three-dimensional (x × y × z) images are acquired from a stack of horizontal section images. The device is in the form of a portable probe. The handle of the probe has a button and a scroll wheel allowing the user to control the imaging modes. Using a supercontinuum laser as a broadband light source and a high numerical microscope objective, an isotropic spatial resolution of â¼1 µm is achieved. The field of view of the three-dimensional images is 1.2 mm × 0.5 mm × 0.5 mm (x × y × z). Images of skin tissues are presented to demonstrate the potential of the technology in dermatology.