Optical Coherence Microscopy: A New Technique for High-Resolution, Non-Invasive Imaging in Bulk Biological Tissues

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.

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.

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.

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.

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.

Towards high resolution imaging of human airways with optical coherence microscopy

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

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.

Optical coherence tomography (OCT) is a non-invasive cross-sectional imaging technique with micrometer resolution. We have been investigating ultrahigh-resolution (UHR)-OCT using supercontinuum. The characteristics of OCT imaging depend on the optical wavelength used. 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 and rat lung tissue. Then we developed UHR-OCT and optical coherence microscopy (OCM) at 1.7 um, which is called as “Optical Window III”. The high-resolution and high-penetration imaging of mouse brain was demonstrated.

Optical Coherence Microscopy (OCM) is a bio-medical low coherence interferometric imaging technique that has become a topic of active research because of its ability to provide accurate, non-invasive cross-sectional images of biological tissue with much greater resolution than the current common technique ultrasound. OCM is a derivative of Optical Coherence Tomography (OCT) that enables greater resolution imposed by the implementation of an optical confocal design involving high numerical aperture (NA) focusing in the sample. The primary setback of OCM, however is the depth dependence of the lateral resolution obtained that arises from the smaller depth of focus of the high NA beam. We propose to overcome this limitation using a dynamic focusing lens design that can achieve quasi-invariant lateral resolution up to 1.5mm depth of skin tissue.

Optical coherence tomography (OCT) is a noninvasive cross-sectional imaging technique with micrometer resolution. The theoretical axial resolution is determined by the center wavelength and bandwidth of the light source, and the wider the bandwidth, the higher the axial resolution. The characteristics of OCT imaging depend on the optical wavelength used. In this paper, we investigated the wavelength dependence of ultrahigh-resolution (UHR) OCT using a supercontinuum for biomedical imaging. Wideband, high-power, low-noise supercontinua (SC) were generated at λ = 0.8, 1.1, 1.3, and 1.7 μ m 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 λ = 0.8–1.0 μ m UHR-OCT. The variation of alveolar volume was estimated using three-dimensional image analysis. Finally, UHR-spectral domain-OCT and optical coherence microscopy at 1.7 μ m were developed, and high-resolution and high-penetration imaging of turbid tissue, especially mouse brain, was demonstrated.

Three dimensional image formation in optical coherence tomography has been investigated theoretically. Imaging can be described by a three-dimensional (3-D) coherent transfer function (CTF), which has contributions from the different wavelength components. From this, two dimensional imaging can be described using the 2-D CTF obtained as a projection of the 3-D CTF. For very low numerical aperture axial imaging results from the limited coherence length of the light source. Interference microscopy results in an optical sectioning property similar to that in confocal microscopy. Thus for intermediate values of numerical aperture, axial imaging is a combination of coherence gating and confocal sectioning, for which a paraxial theory can be used. At very high numerical apertures it is necessary to use a full high aperture theory. These theoretical treatments can be used to model images of known structures, and to estimate expected imaging performance.

.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 tomography (OCT) and confocal laser scanning microscopy (CLSM) are light-based imaging techniques that allow for a visualization of microscopic tissue properties in vivo. Our study was to examine whether they allow for differentiation of inverted papilloma (IP) from nasal polyps (NP). Five cases of IP and NP, respectively, were investigated intraoperatively with OCT and CLSM. Biopsies were taken of the investigated area and were analyzed ex vivo with OCT and CLSM and then underwent HE-staining for standard light microscopy. On OCT images, IP showed the characteristic inverted character of the epithelium, that was thicker with a high degree of variability of thickness compared to the thin and homogenous epithelium of NP. In addition, the characteristic stromal edema of NP could be visualized. On CLSM images, the typical epithelial invaginations of IP appeared as crypts, while in NP the highly organized cylindric epithelium could be visualized. In vivo, OCT acquired images of sufficient quality to visualize these characteristics, while CLSM did not. Our study demonstrates that OCT and CLSM can distinguish IP from NP. Further technical development is required to apply the techniques clinically to guide intranasal biopsies or even to make them dispensable.

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.