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Objective Histopathology is the criterion standard for evaluating cervical squamous intraepithelial neoplasia (dysplasia). In this pilot feasibility study, we examined whether a novel 3-dimensional imaging device using Gabor-domain optical coherence microscopy (GDOCM) could distinguish features of cervical dysplasia comparable with histopathology. Methods A prospective observational pilot study enrolled a small sample of women undergoing loop electrosurgical excision procedure for cervical squamous intraepithelial neoplasia. Fresh ex vivo specimens were imaged with the GDOCM device. Digital images were reviewed by a pathologist who was blinded to the histopathology results. Histopathologic features were then compared with the digital observations. Results Standard histologic features of cervical squamous epithelium and of squamous intraepithelial neoplasia could be observed in GDOCM images. Cervical epithelium, stroma, basement membrane, and squamous papilla could all be identified. Human papillomavirus effects, such as vacuolization and cellular density, were also observed. Conclusions A GDOCM imaging system has the potential to obtain histologic resolution images of the cervix in the evaluation of squamous intraepithelial neoplasia. This pilot study allowed for optimizing the imaging system and paved the way for a future diagnostic accuracy study. The development of this technology could streamline the evaluation of patients at risk for cervical neoplasia.

We quantitatively investigated the image quality in deep tissue imaging with optical coherence microscopy (OCM) in the 1700 nm spectral band, in terms of the signal-to-background ratio (SBR) and lateral resolution. In this work, to demonstrate the benefits of using the 1700 nm spectral band for OCM imaging of brain samples, we compared the imaging quality of OCM en-face images obtained at the same position by using a hybrid 1300 nm/1700 nm spectral domain (SD) OCM system with shared sample and reference arms. By observing a reflective resolution test target through a 1.5 mm-thick tissue phantom, which had a similar scattering coefficient to brain cortex tissue, we confirmed that 1700 nm OCM achieved an SBR about 6-times higher than 1300 nm OCM, although the lateral resolution of the both OCMs was similarly degraded with the increase of the imaging depth. Finally, we also demonstrated high-contrast deep tissue imaging of a mouse brain at a depth up to 1.8 mm by using high-resolution 1700 nm SD-OCM.

The paper presents the study of surface layer structure of two nacre samples, plates of which served as mosaic elements in a subject of decorative art, by spectral optical coherent microscopy method at the wavelength range 1305 ± 75 nm with 10 μm scanning depth resolution. The mesolayers structure with thicknesses of 230 and 360 μm was observed within the B-scan of nacre layer for Haliotis shell. The tomogram of the second nacre sample is characterized by greater nonuniformity of layers’ distribution through the scanning depth, strong scattering of probe radiation, and the microstructure of nacre is visualized in depth up to 1.6 mm. Comparison of the nacre sample layers structure by the optical coherence microscopy method provided for distinguishing their different biological origin. Analysis of the area for mosaic elements clamping to a base of the product is performed and the glue layer thickness is determined in the range from 67 microns to 120 microns.

Dynamic full-field optical coherence microscopy (DFFOCM) was used to characterize the intracellular dynamic activities and cytoskeleton of HeLa cells in different viability states. HeLa cell samples were continuously monitored for 24 hours and compared with histological examination to confirm the cell viability states. The averaged mean frequency and magnitude observed in healthy cells were 4.79±0.5 Hz and 2.44±1.06, respectively. In dead cells, the averaged mean frequency was shifted to 8.57±0.71 Hz, whereas the magnitude was significantly decreased to 0.53±0.25. This cell dynamic activity analysis using DFFOCM is expected to replace conventional time-consuming and biopsies-required histological or biochemical methods.

Significance: Melanocytic nevi represent the most common dermal melanocytic lesions in humans. Nevus is typically diagnosed clinically with the naked eye or with dermoscopy. However, it is essential to identify the type of nevus by invasive biopsy for histopathological examination. The use of noninvasive imaging tools can be used to evaluate the types of nevi to reduce unnecessary excisions of benign entities. Aim: To evaluate the feasibility of using en face and cross-sectional full-field optical coherence tomography (FF-OCT) in differentiation of melanocytic nevi that can facilitate the reduction of unnecessary excisions of benign entities. Approach: Dual-mode Mirau-type FF-OCT for cross-sectional imaging (B-scan) and en face imaging were used to distinguish the types of nevi. Results: Although the B-scan reveals the distribution of melanosomes, users can set a specific depth of the en face image to explore the morphology of surrounding skin cells instantly. According to the locations of nevus nests, the different types of nevi, including junction nevus and compound nevus, can be identified using this dual-mode FF-OCT system. Conclusions: Combining B-scan and en face imaging in vivo FF-OCT enables the examination and navigation of skin tissues in real time and in three dimensions.

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents.

Optical coherence microscopy is an imaging technique capable of generating three-dimensional images of structures in tissue, but with limited functional imaging ability. Fluorescence microscopy offers functional information of cellular activities. The integration of both imaging modalities can enable the evaluation of the impact of structural information on cells activities. To develop an integrated system combining high-resolution optical coherence microscopy (HR-OCM) and dual-channel scanning confocal fluorescence microscopy (DC-SCFM) to enable the co-registration of structural and functional information. Different strategies to enable the simultaneous recording of information, as well as to overcome the focal plane mismatch between both imaging modalities were developed. The system's performances were evaluated in imaging fluorescence microspheres embedded in multi-layer tape and silicone phantom. HR-OCM achieved an axial resolution of 2.4-µm in tissue over an imaging depth of 1-mm. A speed of 250 kHz and a lateral resolution of 2-µm over a field-of-view of 1.1 mm ×1.1 mm were demonstrated with the combined system. DC-SCFM allowed to discriminate between green and red microspheres while HR-OCM enabled their depth localization. The combined system is synergistic in generating structural and functional information of samples, and has the potential to enable new investigations of biological processes in animal models.

The elastic properties of cells are important for many of their functions, however the development of label free noninvasive cellular elastography method is a challenging topic. We present a novel single-cell all-optical coherence elastography method that combines optical tweezers producing mechanical excitation on the cell membrane or organelle and phase-sensitive optical coherence microscopy measuring sample response and determining its mechanical properties. The method allows living cells imaging with a lateral resolution of 0.5 μm and an axial resolution up to 10 nm, making it possible to detect nanometer displacements of the cell organelles and to record the propagation of mechanical wave along the cell membrane in response to optical tweezers excitation. We also demonstrate applicability of the method on single living red blood cells, yeast and cancer cells. The all-optical nature of the method developed makes it a promising and easily applicable tool for studying cellular and subcellular mechanics in vivo.

Cervical cancer remains the fourth most common cause of cancer worldwide and the third leading cause of cancer deaths for women in developing countries. Traditional screening tools, such as human papillomavirus and Pap tests, cannot provide results in real-time and cannot localize suspicious regions. Colposcopy-directed biopsies are invasive in nature and only a few sites of the cervix may be chosen for investigation. A non-invasive, label-free and real-time imaging method with a resolution approaching that of histopathology is desirable for early detection of the disease. Methods: Ultrahigh-resolution optical coherence microscopy (OCM) is an emerging imaging technique used to obtain 3-dimensional (3-D) “optical biopsies” of biological samples with cellular resolution. In this study, 497 3-D OCM datasets from 159 specimens were collected from 92 patients. Results: Distinctive patterns for normal cervix, squamocolumnar junction, ectropion, low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL) and invasive cervical lesions were clearly observed from OCM images, which matched well with corresponding histological slides. OCM images demonstrated a sensitivity of 80% (95% confidence interval, CI, 72%-86%) and a specificity of 89% (95% CI, 84%-93%) for detecting high-risk lesions (HSIL and invasive lesions) when blindly tested by three investigators. A substantial inter-observer agreement was observed (κ=0.627), which showed high diagnostic consistency among three investigators. Conclusion: These results laid the foundation for future non-invasive optical evaluation of cervical tissue in vivo, which could lead to a less invasive and more effective screening and “see-and-treat” strategy for the management of cervical cancer.

We report on the development of a new approach for studying the internal structure of polymer integrated nanophotonics devices using phase-sensitive optical coherence microscopy. Visualization and flaw detection of devices and their internal structure was carried out using the example of coupling gratings and prisms for a miniature Otto configuration with a characteristic gap height of 50-300 nm.

Die häufigste Tumorart des zentralen Nervensystems ist das Gliom. Aufgrund der hohen Variabilität sind die Tumorbeurteilung und die entsprechende Therapie sehr schwierig festzulegen. Daher wird versucht, das chirurgische Vorgehen auf verschiedene Arten zu verbessern. Präoperative Detektionsmethoden wie 5-Aminolävulinsäure (5-ALA) sind im Bereich der niedrig-gradigen Gliome limitiert. Es wird hier die Unterstützung von zusätzlichen Systemen benötigt und auch bereits geprüft. Optische Kohärenztomographie und Fluoreszenzmikroskopie könnten vielversprechende Techniken zur Identifizierung der Intratumorheterogenität sein. In dieser Arbeit wurden diese Methoden eingesetzt, um mit mehreren Messfeldern Unterschiede zwischen Tumorkern und –rand festzustellen. Um diese Anwendungen mit dem Goldstandard zu verifizieren, werden sie mit histopathologischen Standardmethoden verglichen. Aus den histologischen Schnitten wurden Heatmaps für den gesamten Schnitt und mit fünf Messfelder für MIB-1 und HE gewonnen. Die Mikrogefäßdichte wurde anhand eines Scores beurteilt. Die Verwendung von mehreren Messfeldern lieferte dabei zusätzliche Informationen zur genaueren Identifizierung und Abgrenzung der unterschiedlichen Gewebstypen. The most common type of central nervous system tumors is glioma. Due to their high variability, classification and thus an appropriate therapy is very complex. Therefore, attempts are made to improve the surgical procedure in various ways to achieve the best result. Preoperative methods such as 5-aminolevulinic acid (5-ALA) are limited in the area of lower grade gliomas. Here, the support of additional diagnostic tools is needed and examined. Optical coherence tomography and fluorescence microscopy could offer the possibility to visualize intratumor heterogeneity. In this thesis, these methods have been used to distinguish between tumor core and edge by using several fields of view (FOVs) and to determine the tissue structure. To validate these methods against the gold standard, they are compared with standard histopathological markers. Heat maps for the entire histological section and additional five fields of view were obtained for MIB-1 immunohistochemistry and H&E staining. The microvascular proliferation was evaluated by a score. The use of several FOVs provided additional information for the precise identification and delineation of the different tissue types.

We combined phase-sensitive Optical Coherence Microscopy and Optical Tweezers to non-invasively create mechanical excitation in the cell and to detect its response by recording a 3D phase signal.

Gabor-domain optical coherence microscopy (GDOCM) is a high-definition imaging technique leveraging principles of low-coherence interferometry, liquid lens technology, high-speed imaging, and precision scanning. GDOCM achieves isotropic 2 μm resolution in 3D, effectively breaking the cellular resolution limit of optical coherence tomography (OCT). In the ten years since its introduction, GDOCM has been used for cellular imaging in 3D in a number of clinical applications, including dermatology, oncology and ophthalmology, as well as to characterize materials in industrial applications. Future developments will enhance the structural imaging capability of GDOCM by adding functional modalities, such as fluorescence and elastography, by estimating thicknesses on the nano-scale, and by incorporating machine learning techniques.

Был использован метод фазочувствительной оптической когерентной микроскопии (ОКМ) для изучения броуновского движения эритроцита и механического отклика произвольной точки мембраны эритроцита на возмущение другой ее точки, вызванное оптической ловушкой.

To identify the microstructural modification of the corneal layers during the course of the disease, optical technologies have been pushing the boundary of innovation to achieve cellular resolution of deep layers of the cornea. Gabor-domain optical coherence microscopy (GD-OCM), an optical coherence tomography-based technique that can achieve an isotropic of ∼2-μm resolution over a volume of 1 mm×1 mm×1.2 mm, was developed to investigate the microstructural modifications of corneal layers in four common corneal diseases. Since individual layer visualization without cutting through several layers is challenging due to corneal curvature, a flattening algorithm was developed to remove the global curvature of the endothelial layer and display the full view of the endothelium and Descemet’s membrane in single en face images. As a result, GD-OCM revealed the qualitative changes in size and reflectivity of keratocytes in Fuchs endothelial corneal dystrophy (FECD), which varied by the degree of disease. More importantly, elongated shape and hyperactivation characteristics of keratocytes, associated with the early development of guttae, appeared to start in the posterior stroma very early in the disease process and move toward the anterior stroma during disease progression. This work opens a venue into the pathogenesis of FECD.

Cellular traction forces (CTFs) play an integral role in both physiological processes and disease, and are a topic of interest in mechanobiology. Traction force microscopy (TFM) is a family of methods used to quantify CTFs in a variety of settings. State-of-the-art 3D TFM methods typically rely on confocal fluorescence microscopy, which can impose limitations on acquisition speed, volumetric coverage, and temporal sampling or coverage. In this report, we present the first quantitative implementation of a new TFM technique: traction force optical coherence microscopy (TF-OCM). TF-OCM leverages the capabilities of optical coherence microscopy and computational adaptive optics (CAO) to enable the quantitative reconstruction of 3D CTFs in scattering media with minute-scale temporal sampling. We applied TF-OCM to quantify CTFs exerted by isolated NIH-3T3 fibroblasts embedded in Matrigel, with five-minute temporal sampling, using images spanning a 500 × 500 × 500 μm3 field-of-view. Due to the reliance of TF-OCM on computational imaging methods, we have provided extensive discussion of the equations, assumptions, and failure modes of these methods. By providing high-throughput, label-free, volumetric imaging in scattering media, TF-OCM is well-suited to the study of 3D CTF dynamics, and may prove advantageous for the study of large cell collectives, such as the spheroid models prevalent in mechanobiology.

We describe a swept source based phase sensitive optical coherence microscopy (OCM) system for photothermal imaging of gold nanorods (GNR). The phase sensitive OCM system employed in the study has a displacement sensitivity of 0.17 nm to vibrations at single frequencies below 250 Hz. We demonstrate the generation of phase maps and confocal phase images. By displaying the difference between successive confocal phase images, we perform the confocal photothermal imaging of accumulated GNRs behind a glass coverslip and behind the scattering media separately. Compared with two-photon luminescence (TPL) detection techniques reported in literature, the technique in this study has the advantage of a simplified experimental setup and provides a more efficient method for imaging the aggregation of GNR. However, the repeatability performance of this technique suffers due to jitter noise from the swept laser source.

Optical coherence tomography (OCT) is an imaging modality based on the inherent backscattering of light within different tissue types. OCT was introduced in the early 1990s and has since become a standard diagnostic tool in ophthalmology. Images can be acquired non-destructively, in real time and three dimensions at micrometer resolution. Only recently, OCT has been increasingly recognized in other fields such as neuroimaging. Here we present a multimodal imaging approach using a custom-built visible light optical coherence microscope (OCM) combined with a fluorescence imaging mode for the evaluation of different tumor compartments in glioblastoma (GB) samples retrieved during 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. 18 biopsies of ten GB patients were imaged using the visible light OCM system, providing the three-dimensional morphologic structure of the tissue on a cellular level (axial resolution in brain tissue 0.88 µm, penetration depth 100 µm). Attenuation coefficients, i.e., indicators for light penetration and scattering, were calculated for each sample. Tumor-specific contrast enhanced by 5-ALA was evaluated in co-registered images from the fluorescence channel. Samples were ultimately processed for histopathologic work-up and compared to OCM findings. Three different groups of biopsies could be defined based on quantitative 5-ALA fluorescence [normalized between 0 and 1], attenuation [mm-1] and histological H&E stainings: tumor core (n=8; fluorescence [median ± standard deviation]=0.72±0.13, attenuation [median ± standard deviation]=3.9±0.66), infiltration zone (n=6; fluorescence=0.5±0.19, attenuation=4.4±0.5), and adjacent brain parenchyma (n=4; fluorescence=0.3±0.12, attenuation=5.0±0.79). Concurrent increase in fluorescence intensity and cell density was significantly associated with tissue malignancy (tumor core: 3849±1028 nuclei/mm²; brain parenchyma: 1364±236 nuclei/mm². p=0.024). Furthermore, a negative correlation between fluorescence and attenuation coefficient was detected (r=0.51, p=0.032). Aforementioned results suggest that this multimodal imaging setup is a promising approach for non-destructively investigating the three-dimensional morphologic structure at microscopic resolution whilst at the same time leveraging tumor-specific contrast through 5-ALA fluorescence.

Objective: Ultrahigh-resolution optical coherence microscopy (OCM) has recently demonstrated its potential for accurate diagnosis of human cervical diseases. One major challenge for clinical adoption, however, is the steep learning curve clinicians need to overcome to interpret OCM images. Developing an intelligent technique for computer-aided diagnosis (CADx) to accurately interpret OCM images will facilitate clinical adoption of the technology and improve patient care. Methods: 497 high-resolution 3-D OCM volumes (600 cross-sectional images each) were collected from 159 ex vivo specimens of 92 female patients. OCM image features were extracted using a convolutional neural network (CNN) model, concatenated with patient information (e.g., age, HPV results), and classified using a support vector machine classifier. Ten-fold cross-validations were utilized to test the performance of the CADx method in a five-class classification task and a binary classification task. Results: An 88.3 plus or minus 4.9% classification accuracy was achieved for five fine-grained classes of cervical tissue, namely normal, ectropion, low-grade and high-grade squamous intraepithelial lesions (LSIL and HSIL), and cancer. In the binary classification task (low-risk [normal, ectropion and LSIL] vs. high-risk [HSIL and cancer]), the CADx method achieved an area-under-the-curve (AUC) value of 0.959 with an 86.7 plus or minus 11.4% sensitivity and 93.5 plus or minus 3.8% specificity. Conclusion: The proposed deep-learning based CADx method outperformed three human experts. It was also able to identify morphological characteristics in OCM images that were consistent with histopathological interpretations. Significance: Label-free OCM imaging, combined with deep-learning based CADx methods, hold a great promise to be used in clinical settings for the effective screening and diagnosis of cervical diseases., Comment: 9 pages, 5 figures, and 2 tables

Optical coherence tomography (OCT) is an optical technique that uses backscattered light to highlight intrinsic structure, and when applied to brain tissue, it can resolve cortical layers and fiber bundles. Optical coherence microscopy (OCM) is higher resolution (i.e., 1.25 μm) and is capable of detecting neurons. In a previous report, we compared the correspondence of OCM acquired imaging of neurons with traditional Nissl stained histology in entorhinal cortex layer II. In the current method-oriented study, we aimed to determine the colocalization success rate between OCM and Nissl in other brain cortical areas with different laminar arrangements and cell packing density. We focused on two additional cortical areas: medial prefrontal, pre-genual Brodmann area (BA) 32 and lateral temporal BA 21. We present the data as colocalization matrices and as quantitative percentages. The overall average colocalization in OCM compared to Nissl was 67% for BA 32 (47% for Nissl colocalization) and 60% for BA 21 (52% for Nissl colocalization), but with a large variability across cases and layers. One source of variability and confounds could be ascribed to an obscuring effect from large and dense intracortical fiber bundles. Other technical challenges, including obstacles inherent to human brain tissue, are discussed. Despite limitations, OCM is a promising semi-high throughput tool for demonstrating detail at the neuronal level, and, with further development, has distinct potential for the automatic acquisition of large databases as are required for the human brain.

Gabor-domain optical coherence microscopy (GDOCM) demonstrated in vivo corneal imaging with cellular resolution and differentiation in mice over a field of view of 1 mm2. Contact and non-contact imaging was conducted on six healthy and six hyperglycemic C57BL/6J mice. Cellular resolution in the 3D GDOCM images was achieved after motion correction. Corneal nerve fibers were traced and their lengths and branches calculated. Noncontact, label-free imaging of corneal nerves has clinical utility in health and disease, and in transplant evaluation. To the authors’ knowledge, this is the first report of in vivo 3D corneal imaging in mice with the capability to resolve nerve fibers using a non-contact imaging modality.

The surface layers microstructure of biological tissue on the example of plant fruit exocarp was investigated by spectral optical coherence microscopy with tunable wavelength in the 1305  75 nm range and by the method of correlated optical coherence microscopy at the mean wavelength value equal to 940 nm. The experiments were performed for the intact and defect structures, and calculation of the surface profile of exocarp was performed.

This paper explores the potential of optical coherence microscopy (OCM) for the in situ monitoring of biofilm growth. The quantitative imaging of the early developmental biology of a representative biofilm, Klebsiella pneumonia (KP-1), was performed using a swept source-based Fourier domain OCM system. The growth dynamics of the KP-1 biofilms and their transient response under perturbation was investigated using the enface visualization of microcolonies and their spatial localization. Furthermore, the optical density (OD) and planar density of the biofilms are calculated using an OCM technique and compared with OD and colony forming units measured using standard procedures via the sampling of the flow-cell effluent. MOE (Min. of Education, S’pore) EDB (Economic Devt. Board, S’pore) Published version

We used Optical Coherence Microscopy (OCM) to monitor structural and functional changes due to ischemic stroke in small animals brains in vivo. To obtain lateral resolution of 2.2 μm over the range of 600 μm we used extended focus configuration of OCM instrument involving Bessel beam. It provided access to detailed 3D information about the changes in brain vascular system up to the level of capillaries across I and II/III layers of neocortex. We used photothrombotic stroke model involving photoactive application of rose bengal to assure minimal invasiveness of the procedure and precise localization of the clot distribution center. We present the comparative analysis involving structural and angiographic maps of the stroke-affected brain enabling in-depth insight to the process of development of the disorder.

We demonstrate label-free imaging of cerebral β-amyloidosisex vivoand in a living mouse model of Alzheimer's disease using extended-focus Fourier domain optical coherence microscopy (xfOCM). xfOCM provides 3D, high-resolution images of individual β-amyloid plaques in the brain parenchyma and vasculature and requires no staining of the Alzheimeric sample under investigation. xfOCM also opens the possibility to perform minimally invasive studies of β-amyloid pathologyin vivo, without the use of labeling methods, which potentially confound experimental findings.

We demonstrate full-field optical coherence microscopy (OCM) using an ultrathin forward-imaging short multimode fiber (SMMF) probe with a core diameter of 50 &mu, m, outer diameter of 125 &mu, m, and length of 7.4 mm, which is a typical graded-index multimode fiber used for optical communications. The axial and lateral resolutions were measured to be 2.14 &mu, m and 2.3 &mu, m, respectively. By inserting the SMMF 4 mm into the cortex of an in vivo rat brain, scanning was performed to a depth of 147 &mu, m from the SMMF facet with a field of view of 47 &mu, m. Three-dimensional (3D) OCM images were obtained at depths ranging from approximately 20 &mu, m to 90 &mu, m. Based on the morphological information of the resliced 3D images and the dependence of the integration of the OCM image signal on the insertion length, the obtained 3D information of nerve fibers has been presented.