Integration of line-field confocal optical coherence tomography and in situ microenvironmental mapping to investigate the living microenvironment of reconstructed human skin and melanoma models.

BACKGROUND: In tissue engineering, real-time monitoring of tumors and of the dynamics of the microenvironment within in vitro models has traditionally been hindered by the need to harvest the cultures to obtain material to analyze. Line-field confocal optical coherence tomography (LC-OCT) has proven to be useful in evaluating in vivo skin conditions, including melanoma, by capturing dynamic, three-dimensional (3D) information without the need for invasive procedures, such as biopsies. Additionally, the M-Duo Technology® developed by IMcoMET presents a unique opportunity for continuous in situ biomarker sampling, providing insights into local cellular behavior and interactions. OBJECTIVE: This study aimed to validate the non-destructive mapping capabilities of two advanced methodologies (LC-OCT by DAMAE Medical and M-Duo Technology® by IMcoMET) to investigate the living microenvironment of in vitro reconstructed human skin (RhS) and melanoma-RhS (Mel-RhS). METHODS: LC-OCT and M-Duo Technology® were compared to conventional analysis of the RhS and Mel-RhS microenvironments. RESULTS: LC-OCT successfully visualized the distinct layers of the epidermis and tumor structures within the Mel-RhS, identifying keratinocytes, melanocytes, tumor nests, and fibroblasts. The M-Duo Technology® revealed differences in in situ cytokine (IL-6) and chemokine (CCL2, CXCL10, and IL-8) secretion between Mel-RhS and the control RhS. Notably, such differences were not detected through conventional investigation of secreted proteins in culture supernatants. CONCLUSION: The combination of LC-OCT's high-resolution imaging and M-Duo Technology®'s in situ microenvironmental mapping has the potential to provide a synergistic platform for non-invasive, real-time analysis, allowing for prolonged observation of processes within Mel-RhS models without the need for culture disruption.

Line-Field Confocal Optical Coherence Tomography (LC-OCT) for Skin Imaging in Dermatology.

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.

Mirau-based line-field confocal optical coherence tomography for three-dimensional high-resolution skin imaging.

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 ∼1.5 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 ∼2 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.