Mirau-based line-field confocal optical coherence tomography.

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.

Line-field confocal time-domain optical coherence tomography with dynamic focusing.

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 ∼500 µm.

High-resolution full-field optical coherence microscopy using a broadband light-emitting diode.

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.

Wide-field, full-field optical coherence microscopy for high-axial-resolution phase and amplitude imaging.

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 ∼80 dB through the entire field of view are obtained and applied to biological specimens.

Line-field confocal optical coherence tomography based on tandem interferometry with a focus-tunable lens.

This article introduces an innovative line-field confocal optical coherence tomography (LC-OCT) system based on tandem interferometry, featuring a focus-tunable lens for dynamic focusing. The principle of tandem interferometry is first recalled, and an analytical expression of the interferometric signal detected is established in order to identify the influence of key experimental parameters. The LC-OCT system is based on a Linnik-type imaging interferometer with a focus-tunable lens for focus scanning, coupled to a Michelson-type compensating interferometer using a piezoelectric linear translation stage for coherence plane scanning. The system achieves axial and lateral image resolutions of approximately 1 µm over the entire imaging depth (400 µm), in line with conventional LC-OCT. Vertical section images (B-scans) of skin acquired at 14.3 fps reveal distinguishable structures within the epidermis and dermis. Using refocusing and stitching, images of a tissue phantom were obtained with an imaging depth superior to 1.4 mm. The system holds promise for LC-OCT miniaturization, along with enhanced imaging speed and extended imaging depth.

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): principles and practical use.

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.

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.

Co-localized line-field confocal optical coherence tomography and confocal Raman microspectroscopy for three-dimensional high-resolution morphological and molecular characterization of skin tissues ex vivo.

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.

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.

Dual-mode line-field confocal optical coherence tomography for ultrahigh-resolution vertical and horizontal section imaging of human skin in vivo.

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 ∼ 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.

Line-field confocal optical coherence tomography for three-dimensional skin imaging.

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 ∼1 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.

A compact high-speed full-field optical coherence microscope for high-resolution in vivo skin imaging.

A compact high-speed full-field optical coherence microscope has been developed for high-resolution in vivo imaging of biological tissues. The interferometer, in the Linnik configuration, has a size of 11 × 11 × 5 cm3 and a weight of 210 g. Full-field illumination with low-coherence light is achieved with a high-brightness broadband light-emitting diode. High-speed full-field detection is achieved by using part of the image sensor of a high-dynamic range CMOS camera. En face tomographic images are acquired at a rate of 50 Hz, with an integration time of 0.9 ms. The image spatial resolution is 0.9 µm × 1.2 µm (axial × transverse), over a field of view of 245 × 245 µm2 . Images of human skin, revealing in-depth cellular-level structures, were obtained in vivo and in real-time without the need for stabilization of the subject. The system can image larger fields, up to 1 × 1 mm2 , but at a reduced depth.