Invariant high resolution optical skin imaging

Optical coherence microscopy (OCM) is a high-resolution imaging technique based on optical coherence tomography and confocal microscopy. The recent studies on OCM operating at 800-1300 nm spectral region have shown that OCM enables to visualize micrometer- or sub-micrometer-scale structures of animal tissues. Although OCMs offers such high-resolution label-free imaging capability of animal tissues, the imaging depth was restricted by multiple light scattering and light absorption of water in samples. Here, for high-resolution deep-tissue imaging, we developed an OCM in the 1700-nm spectral band by using a supercontinuum (SC) source with a Gaussian-like spectral shape in the wavelength region. Recently, it has been reported that the 1700-nm spectral band is a promising choice for enhancing the imaging depth in the observation of turbid scattering tissues because of the low attenuation coefficient of light. In this study, to clarify that the 1700-nm OCM has a potential to realize the enhanced imaging depth, we compared the attenuation of the signal-to-noise ratio between the 1700-nm and 1300-nm OCM imaging of a mouse brain under the same signal detection sensitivity condition. The result shows that the 1700-nm OCM enables us to achieve the enhanced imaging depth. In this 1700-nm OCM, we also confirmed that the lateral resolution of 1.3 µm and axial resolution of 2.8 µm in tissue were achieved.

To investigate optical coherence microscopy (OCM) imaging features and the application value of these high-resolution images for identifying endocervical canal lesions (ECLs), which is a clinical dilemma in cervical cancer screening programs. In total, 520 OCM images were obtained by scanning the cervical canal lesions with an ultra-high-resolution OCM system (204 specimens from 73 patients). The OCM morphologic characteristics of ECLs were observed and summarized, and then 3 researchers performed a diagnostic test of OCM images of cervical canal lesions. The accuracy, sensitivity, specificity, positive predictive value, negative predictive value, 95% confidence interval of each parameter, and interinvestigator agreement (κ) were calculated. Normal endocervix, cysts, squamous metaplasia, high-grade squamous intraepithelial lesions involving glands, and invasive carcinoma had distinct OCM characteristics, which correlated well with corresponding H&E histologic sections. The accuracy, sensitivity, and specificity of the 3 researchers were 90.6%, 89.3% (95% CI, 86.5%-91.7%) and 91.6% (95% CI, 89.2%-93.5%), respectively. The positive predictive value was 90.1% (95% CI, 87.3%-92.4%), and the negative predictive value was 90.9% (95% CI, 88.5%-92.9%), with almost perfect agreement (κ = 0.874). The application of the OCM system in cervical canal lesions is feasible and could help improve detection of occult ECLs in cervical cancer screening programs. This study lays the foundation for further research on OCM in cervical canal lesions in vivo, which also has a potential impact on projecting pathologic evaluation beyond what is currently possible, perhaps globally.

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.

.We present a multimodal visible light optical coherence microscopy (OCM) and fluorescence imaging (FI) setup. Specification and phantom measurements were performed to characterize the system. Two applications in neuroimaging were investigated. First, curcumin-stained brain slices of a mouse model of Alzheimer’s disease were examined. Amyloid-beta plaques were identified based on the fluorescence of curcumin, and coregistered morphological images of the brain tissue were provided by the OCM channel. Second, human brain tumor biopsies retrieved intraoperatively were imaged prior to conventional neuropathologic work-up. OCM revealed the three-dimensional structure of the brain parenchyma, and FI added the tumor tissue-specific contrast. Attenuation coefficients computed from the OCM data and the florescence intensity values were analyzed and showed a statistically significant difference for 5-aminolevulinic acid (5-ALA)-positive and -negative brain tissues. OCM findings correlated well with malignant hot spots within brain tumor biopsies upon histopathology. The combination of OCM and FI seems to be a promising optical imaging modality providing complementary contrast for applications in the field of neuroimaging.

Optical coherence microscopy (OCM) is a novel technique complementary to optical coherence tomography (OCT) which combines low-coherence interferometry with confocal microscopy to achieve micron-scale resolution imaging in highly scattering media. OCM may be implemented using a single-mode fiber-optic low-coherence interferometer (See Fig. 1). A high numerical aperture objective is used to focus sample-arm light into the specimen, and the reference arm length of the interferometer is adjusted to match the sample arm focal plane optical depth. The sample arm of the interferometer comprises a scanning confocal microscope, in which either the sample or the probe beam is laterally scanned in a raster pattern, and the optical fiber acts as a single-mode confocal aperture for combined light illumination and collection. The reference arm length of the interferometer establishes the depth position of an interferometric “coherence gate” in the sample, from which backscattered light is preferentially collected. Initial studies of OCM in scattering phantoms have demonstrated that this technique provides increased optical sectioning depth compared to confocal microscopy alone.

Optical coherence microscopy (OCM) has unique advantages of high-resolution volumetric imaging without relying on exogenous labels or dyes. It combines the coherence-gated depth discrimination of optical coherence tomography (OCT) with the high lateral resolution of confocal microscopy, offering an excellent balance between the resolutions and imaging depth. However, as the lateral resolution becomes higher, the imaging depth of OCM decreases and its three-dimensional imaging capability is greatly degraded. To overcome this limitation, we used amplitude apodization to create quasi-Bessel beam illumination in order to extend the depth of focus. The lateral and axial resolutions of our OCM system were measured to be 1.6 μm and 2.9 μm in tissue. The imaging depth was extended by ~3.0X (~100 μm) beyond that of the standard Gaussian beam OCM. Using zebrafish embryos as a test system, we demonstrate extendedfocus OCM for structural imaging studies, which revealed the detailed anatomy deep in embryos.

Towards high resolution imaging of human airways with optical coherence microscopy

Optical coherence tomography (OCT) is a non-invasive cross-sectional imaging technique with micrometer resolution. OCT is useful, non-invasive imaging technique of the internal structure, and it has been applied in many fields, especially medicine and industry. The theoretical axial resolution is determined by the center wavelength and bandwidth of the light source, and the wider the bandwidth is, the higher the axial resolution is. Supercontinuum is the high-power, ultrawideband light source. We have been investigating ultrahigh-resolution (UHR)-OCT using supercontinuum. The characteristics of OCT imaging depend on the optical wavelength used. In this talk, we report our recent work of the wavelength dependence of UHR-OCT using a supercontinuum for biomedical imaging. In order to investigate the wavelength dependence of UHR-OCT, the wideband, high-power, low-noise supercontinua were generated at wavelengths of 0.8, 1.1, 1.3, and 1.7 um based on ultrashort pulses and nonlinear fibers. The wavelength dependence of OCT imaging was examined quantitatively using biological phantoms. Ultrahigh-resolution imaging of a rat lung was demonstrated with wavelengths of 0.8 – 1.0 um UHR-OCT. The variation of alveolar volume was estimated using 3D image analysis. We also developed UHR-spectral domain-OCT and optical coherence microscopy (OCM) at 1.7 um. The high-resolution and high-penetration imaging of turbid tissue, especially mouse brain, was demonstrated. The wavelength dependence of OCM was also discussed in terms of mouse brain imaging.

Optical coherence microscopy (OCM) is a novel approach to fluorophore-free 3D live imaging of cells, particularly mammalian oocytes and embryos. It allows for 3D high-resolution visualization of the intracellular architecture: nuclei with nucleoli, metaphase spindles, and networks of membranous structures. Moreover, as it is compatible with time-lapse imaging, it enables monitoring and quantitative analysis of the dynamic behavior of these organelles over time. Importantly, OCM, when imaging settings are properly optimized, is safe for oocytes and embryos and does not negatively affect their developmental capabilities. Therefore, OCM is an interesting alternative to currently used imaging techniques, not only in basic research but also in clinical applications. In the present chapter, we describe the main principles of spectral OCM and show how this technique can be applied to visualize mouse oocytes.

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Fluorescence-guided surgery is a state-of-the-art approach for intraoperative imaging during neurosurgical removal of tumor tissue. While the visualization of high-grade gliomas is reliable, lower grade glioma often lack visible fluorescence signals. Here, we present a hybrid prototype combining visible light optical coherence microscopy (OCM) and high-resolution fluorescence imaging for assessment of brain tumor samples acquired by 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. OCM provides high-resolution information of the inherent tissue scattering and absorption properties of tissue. We here explore quantitative attenuation coefficients derived from volumetric OCM intensity data and quantitative high-resolution 5-ALA fluorescence as potential biomarkers for tissue malignancy including otherwise difficult-to-assess low-grade glioma. We validate our findings against the gold standard histology and use attenuation and fluorescence intensity measures to differentiate between tumor core, infiltrative zone and adjacent brain tissue. Using large field-of-view scans acquired by a near-infrared swept-source optical coherence tomography setup, we provide initial assessments of tumor heterogeneity. Finally, we use cross-sectional OCM images to train a convolutional neural network that discriminates tumor from non-tumor tissue with an accuracy of 97%. Collectively, the present hybrid approach offers potential to translate into an in vivo imaging setup for substantially improved intraoperative guidance of brain tumor surgeries.

A multimodal technique for inspection of microscopic objects by means of wideband optical microscopy, spectral microscopy, and optical coherence microscopy is described, implemented, and tested. The key feature is the spectral selection of light in the output arm of an interferometer with use of the specialized imaging acousto-optical tunable filter. In this filter, two interfering optical beams are diffracted via the same ultrasound wave without destruction of interference image structure. The basic requirements for the acousto-optical tunable filter are defined, and mathematical formulas for calculation of its parameters are derived. Theoretical estimation of the achievable accuracy of the 3D image reconstruction is presented and experimental proofs are given. It is demonstrated that spectral imaging can also be accompanied by measurement of the quantitative reflectance spectra. Examples of inspection of optically transparent and nontransparent samples demonstrate the applicability of the technique.

OCT-A is becoming more popular in recent years and there is a high demand to improve the quality of angiograms as well as to extract quantitative information. We applied various processing methods for microvasculature enhancement like Hessian-Frangi to a data set obtained with Bessel and Gaussian OCT systems. We used angiogenesis, fractal and multifractal analysis to extract more quantitative information. We applied the processing methods for healthy, stroke, tumor progression and the results of enhanced processing and quantitative analysis for those are presented in this letter. Full Text: PDF ReferencesJ. G. Fujimoto, C. Pitris, S. A. Boppart, and M. E. Brezinski, "Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos", Neoplasia (New York, NY), 2000, 2(1-2):9-25 CrossRef O. Liba, E. D. SoRelle, D. Sen, and A. de la Zerda, "Contrast-enhanced optical coherence tomography with picomolar sensitivity for functional in vivo imaging", Sci Rep. 2016, 6(1):23337. CrossRef O Liba, M. D. Lew, E. D. SoRelle, et al., "Speckle-modulating optical coherence tomography in living mice and humans", Nat Commun. 2017, 8:15845. CrossRef K. Karnowski, A. Ajduk, B. Wieloch, et al., "Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos", Sci Rep. 2017, 7(1):4165. CrossRef V. J. Srinivasan, S. Sakadžić, I. Gorczynska, et al., "Quantitative cerebral blood flow with Optical Coherence Tomography", Opt Express. 2010, 18(3):2477. CrossRef S. Tamborski, H. C. Lyu, H. Dolezyczek, et al. Extended-focus optical coherence microscopy for high-resolution imaging of the murine brain. Biomed Opt Express. 2016, 7(11):4400-4414. CrossRef A. F. Frangi, W. J. Niessen, K. L. Vincken, and M. A. Viergever, "Multiscale vessel enhancement filtering", Lect Notes Comput Sc. 1998;1496:130–137 CrossRef A. A. Ucuzian, A. A. Gassman, A. T. East, and H. P. Greisler , Journal of burn care & research: official publication of the American Burn Association 2010 , 31(1):158. CrossRef R. Lopes, and Betrouni, N. (2009). "Fractal and multifractal analysis: A review", Med. Image Anal. 13, 634–649. CrossRef J. W. Baish and R. K. Jain., "Correspondence re: J. W. Baish and R. K. Jain, Fractals and Cancer. Cancer Res., 60: 3683-3688, 2000.", Cancer Res. 2000, Jul 15, 60(14):3683-8. DirectLink

Along with high longitudinal resolution of optical coherence tomography, confocal optical coherence microscopy (OCM) provides high transversal resolution due to relatively high numerical apertures. However, the presence of relatively high numerical apertures leads to limited depth of field, which reduces the speed of OCM and decreases the advantage of Fourier domain detection. In this paper we propose a numerical processing technique for three-dimensional image reconstruction in Fourier domain OCM. It takes into account not only the defocus of different parts of imaged volumetric sample, but also the effects of upper layers’ refractive index on imaging the sample inner structure. Besides providing sharp coherence gated imaging, this technique also allows for determining both the geometrical thickness and refractive index of the sample layers.

Optical coherence microscopy (OCM) is a non-destructive 3-dimentional (3D) imaging technique with micrometer-scale resolution, which is based on optical coherence tomography (OCT) and confocal microscopy [1-3]. Recently, it has been demonstrated that the OCT imaging depth is improved by using 1700 nm spectral band, so called “optical window 3”, due to the lower light absorption and a local minimum of water absorption in the wavelength region [4-8]. In our group, we developed a supercontinuum (SC) laser source at the 1700-nm wavelength region and demonstrated that 1700-nm OCT allows us to visualize the structures of mouse brain at a depth up to 1.7 mm [9-11]. We also successfully demonstrated 3D highresolution visualization of myelinated axons in a mouse brain with 1700-nm OCM, which we developed based on a time-domain low-coherence interferometer [12]. In this presentation, we report high-resolution spectral-domain OCM (SD-OCM) at the 1700-nm spectral band for improving the imaging acquisition speed of 1700-nm OCM.