Full-field Optical Coherence Tomography Research Articles

Full-field optical coherence tomography for the analysis of fresh unstained human lobectomy specimens

Background: Full-field optical coherence tomography (FFOCT) is a real-time imaging technique that generates high-resolution three-dimensional tomographic images from unprocessed and unstained tissues. Lack of tissue processing and associated artifacts, along with the ability to generate large-field images quickly, warrants its exploration as an alternative diagnostic tool. Materials and Methods: One section each from the tumor and from adjacent non-neoplastic tissue was collected from 13 human lobectomy specimens. They were imaged fresh with FFOCT and then submitted for routine histopathology. Two blinded pathologists independently rendered diagnoses based on FFOCT images. Results: Normal lung architecture (alveoli, bronchi, pleura and blood vessels) was readily identified with FFOCT. Using FFOCT images alone, the study pathologists were able to correctly identify all tumor specimens and in many cases, the histological subtype of tumor (e.g., adenocarcinomas with various patterns). However, benign diagnosis was provided with high confidence in roughly half the tumor-free specimens (others were diagnosed as equivocal or false positive). Further analysis of these images revealed two major confounding features: (a) Extensive lung collapse and (b) presence of smoker’s macrophages. On a closer inspection, however, the smoker’s macrophages could often be identified as distinct from tumor cells based on their relative location in the alveoli, size and presence of anthracosis. We posit that greater pathologist experience, complemented with morphometric analysis and color-coding of image components, may help minimize the contribution of these confounders in the future. Conclusion: Our study provides evidence for the potential utility of FFOCT in identifying and differentiating lung tumors from non-neoplastic lung tissue. We foresee its potential as an adjunct to intra-surgical frozen section analysis for margin assessment, especially in limited lung resections.

Imaging of non-tumorous and tumorous human brain tissues with full-field optical coherence tomography

A prospective study was performed on neurosurgical samples from 18 patients to evaluate the use of full-field optical coherence tomography (FF-OCT) in brain tumor diagnosis.FF-OCT captures en face slices of tissue samples at 1 μm resolution in 3D to a penetration depth of around 200 μm. A 1 cm2 specimen is scanned at a single depth and processed in about 5 min. This rapid imaging process is non-invasive and requires neither contrast agent injection nor tissue preparation, which makes it particularly well suited to medical imaging applications.Temporal chronic epileptic parenchyma and brain tumors such as meningiomas, low-grade and high-grade gliomas, and choroid plexus papilloma were imaged. A subpopulation of neurons, myelin fibers and CNS vasculature were clearly identified. Cortex could be discriminated from white matter, but individual glial cells such as astrocytes (normal or reactive) or oligodendrocytes were not observable.This study reports for the first time on the feasibility of using FF-OCT in a real-time manner as a label-free non-invasive imaging technique in an intraoperative neurosurgical clinical setting to assess tumorous glial and epileptic margins.

Large Field, High Resolution Full-Field Optical Coherence Tomography: A Pre-clinical Study of Human Breast Tissue and Cancer Assessment

We present a benchmark pilot study in which high-resolution Full-Field Optical Coherence Tomography (FF-OCT) was used to image human breast tissue and is evaluated to assess its ability to aid the pathologist’s management of intra-operative diagnoses. FF-OCT imaging safety was investigated and agreement between FF-OCT and routinely prepared histopathological images was evaluated. The compact setup used for this study provides 1 µm3 resolution and 200 µm imaging depth, and a 2.25 cm2 specimen is scanned in about 7 minutes. 75 breast specimens were imaged from 22 patients (21 women, 1 man) with a mean age of 58 (range: 25-83). Pathologists blind diagnosed normal/benign or malignant tissue based on FF-OCT images alone, diagnosis from histopathology followed for comparison. The contrast in the FF-OCT images is generated by intrinsic tissue scattering properties, meaning that no tissue staining or preparation is required. Major architectural features and tissue structures of benign breast tissue, including adipocytes, fibrous stroma, lobules and ducts were characterized. Subsequently, features resulting from pathological modification were characterized and a diagnosis decision tree was developed. Using FF-OCT images, two breast pathologists were able to distinguish normal/benign tissue from lesional with a sensitivity of 94% and 90%, and specificity of 75% and 79% respectively.

Enhancement of the resolution of full-field optical coherence tomography by using a colour image sensor

The influence of white balance in a colour image detector on the resolution of a full-field optical coherence tomograph (FFOCT) is studied. The change in the interference pulse width depending on the white balance tuning is estimated in the cases of a thermal radiation source (incandescent lamp) and a white light emitting diode. It is shown that by tuning white balance of the detector in a certain range, the FFOCT resolution can be increased by 20% as compared to the resolution, attained with the use of a monochrome detector.

Understanding three-dimensional spatial relationship between the mouse second polar body and first cleavage plane with full-field optical coherence tomography

The morphogenetic relationship between early patterning and polarity formation is of fundamental interest and remains a controversial issue in preimplantation embryonic development. We use a label-free three-dimensional (3-D) imaging technique of full-field optical coherence tomography (FF-OCT) successfully for the first time to study the dynamics of developmental processes in mouse preimplantation lives. Label-free 3-D subcellular time-lapse images are demonstrated to investigate 3-D spatial relationship between the second polar body (2PB) and the first cleavage plane. By using FF-OCT together with quantitative study, we show that only 25% of the predicted first cleavage planes, defined by the apposing plane of two pronuclei, pass through the 2PB. Also only 27% of the real cleavage planes pass through the 2PB. These results suggest that the 2PB is not a convincing spatial cue for the event of the first cleavage. Our studies demonstrate the feasibility of FF-OCT in providing new insights and potential breakthroughs to the controversial issues of early patterning and polarity in mammalian developmental biology.

Analysis of interferograms of multi-layered biological samples obtained from full field optical coherence tomography systems

Several methods were developed in the past to analyze interferograms produced by optical coherence tomography, and successfully applied to simulated or animated samples. However, these techniques do not cope with noisy and distorted interferograms from biological tissues. In this paper, known techniques, including the fast Fourier transform and several variations of the continuous wavelet transform, were employed to analyze the interferogram data. However, to cope with the difficulties in biological data, pre- and post-processing procedures and adaptive thresholding were developed to provide stability and robustness. Additionally, three-dimensional structural models of the biological samples were constructed, and revealed information like the number and locations of interfaces, the layer thickness and pattern, and abnormalities.

White-light full-field OCT resolution improvement by image sensor colour balance adjustment: numerical simulation

The possibility of improving white-light full-field optical coherence tomography (OCT) resolution by image sensor colour balance tuning is shown numerically. We calculated the full-width at half-maximum (FWHM) of a coherence pulse registered by a silicon colour image sensor under various colour balance settings. The calculations were made for both a halogen lamp and white LED sources. The results show that the interference pulse width can be reduced by the proper choice of colour balance coefficients. The reduction is up to 18%, as compared with a colour image sensor with regular settings, and up to 20%, as compared with a monochrome sensor.

Colour Sensor White Balance Influence on White-Light Interferometer Resolution

Impact of a sensor color balance on white-light fullfieldoptical coherence tomography (FF-OCT)resolution is investigated in the paper. Full width athalf magnitude (FWHM) of a coherence pulse wascalculated for various white balance (WB) settings.Simulated dependence of FWHM on WB of asensor and experimental data are shown in thepaper. The minimum interference pulse width canbe achieved by the proper color balance coefficientschoice. And in this case the pulse is narrower thaneither one registered by colour image sensor withWB based on light source emission spectrum or bymonochrome image sensor.

Spatial and temporal coherence effects in interference microscopy and full‐field optical coherence tomography

AbstractLow‐coherence optical microscopy or optical coherence microscopy uses light with short coherence length. The well‐known case is: “white‐light interferometry”, which became recently more known as: “optical coherence tomography”. However, when lenses and microscope objectives are used to create interferometric images, in what is known classically as “interference microscopy” or today as “full‐field optical coherence tomography” the spatial coherence starts to play a critical role. In this article the coherence effects in low‐coherence optical microscopy are reviewed. As this technology is becoming increasingly publicized due to its importance in three‐dimensional imaging, particularly of scattering biological media and optical metrology, the understanding of the fundamental physics behind it is essential. The interplay between longitudinal spatial coherence and temporal coherence and the effects associated with them are discussed in detail particularly when high numerical apertures are used. An important conclusion of this study is that a high‐contrast, high‐resolution system for imaging of multilayered samples is the one that uses narrowband illumination and high‐NA objectives with an index‐matching fluid. Such a system, when combined with frequency‐domain operation, can reveal nearly real‐time three‐dimensional images, and is thus competitive with confocal microscopy.

Efficient holoscopy image reconstruction

Holoscopy is a tomographic imaging technique that combines digital holography and Fourier-domain optical coherence tomography (OCT) to gain tomograms with diffraction limited resolution and uniform sensitivity over several Rayleigh lengths. The lateral image information is calculated from the spatial interference pattern formed by light scattered from the sample and a reference beam. The depth information is obtained from the spectral dependence of the recorded digital holograms. Numerous digital holograms are acquired at different wavelengths and then reconstructed for a common plane in the sample. Afterwards standard Fourier-domain OCT signal processing achieves depth discrimination. Here we describe and demonstrate an optimized data reconstruction algorithm for holoscopy which is related to the inverse scattering reconstruction of wavelength-scanned full-field optical coherence tomography data. Instead of calculating a regularized pseudoinverse of the forward operator, the recorded optical fields are propagated back into the sample volume. In one processing step the high frequency components of the scattering potential are reconstructed on a non-equidistant grid in three-dimensional spatial frequency space. A Fourier transform yields an OCT equivalent image of the object structure. In contrast to the original holoscopy reconstruction with backpropagation and Fourier transform with respect to the wavenumber, the required processing time does neither depend on the confocal parameter nor on the depth of the volume. For an imaging NA of 0.14, the processing time was decreased by a factor of 15, at higher NA the gain in reconstruction speed may reach two orders of magnitude.

Tomographic imaging of a suspending single live cell using optical tweezer-combined full-field optical coherence tomography

We propose a label-free depth-resolved tomographic scheme for imaging a single live cell in fluid. This approach utilizes a modified time-domain full-field optical coherence tomography (FF-OCT) system combined with an optical tweezer technique. The optical trap for holding a moving specimen is made by tightly focusing a 1064 nm Q-switching pulsed laser beam with a 1.0 NA microscope objective in the sample arm of the FF-OCT part. By cosharing the probe for both systems, the optical actions of trapping and cellular resolution tomographic imaging could be achieved simultaneously. Feasibility of the combined system is demonstrated by imaging micron-sized polystyrene beads and a living suspension cell in medium.

Simultaneous shape measurement and layers detection by means of low coherence interferometry and optical coherence tomography

In this paper authors show the application of low coherence interferometry and optical coherence tomography with non-Gaussian light source, which are applicable in simultaneous measurement of micromembrane and detection of layers deposited on silicon wafer. The exemplary measurement results obtained with laboratory setup are presented. Full Text: PDF References D. Huang, et al. "Optical coherence tomography", Science, Vol. 254, 1178 (1991). CrossRef A. Dubois, L. Vabre, A.C. Boccara and E.l Beaurepaire, "High-Resolution Full-Field Optical Coherence Tomography with a Linnik Microscope", Appl. Opt. Vol. 41, 805 (2002). CrossRef J.W. Goodman, Statistical Optics (John Wiley & Sons 1985). A. Harasaki, J. Schmit and J.C. Wyant, "Offset of Coherent Envelope Position Due to Phase Change on Reflection", Appl. Opt. Vol. 40, 2101 (2001). CrossRef G. Kieran, J. Larkin, "Efficient nonlinear algorithm for envelope detection in white light interferometry", J. Opt. Soc. Am. A, Vol. 13, 832 (1996). CrossRef A. Hirabayashi, H. Ogawa and K. Kitagawa, "Fast Surface Profiler by White-Light Interferometry by use of a New Algorithm Based on Sampling Theory", Appl. Opt. Vol. 41, 4876 (2002). CrossRef J. Plucinski et al., "Optical low-coherence interferometry for selected technical applications", Bulletin of the Polish Academy of Sciences, Vol. 56, 155 (2008). DirectLink A. Pakula, L. Salbut, Fringe 2009, Springer, 358 (2009). S. Chen, A.W. Palmer, "Fringe order identification in optical fibre white-light interferometry using centroid algorithm method", Electronics Lett., Vol. 28, 553, (1992). CrossRef A. Sabac, C. Gorecki and M. Jozwkik, "Design, testing, and calibration of an integrated Mach-Zehnder-based optical read-out architecture for MEMS characterization", Proc SPIE, VOL. 5458, 141 (2004). DirectLink

Label-free subcellular 3D live imaging of preimplantation mouse embryos with full-field optical coherence tomography

Early patterning and polarity is of fundamental interest in preimplantation embryonic development. Label-free subcellular 3D live imaging is very helpful to its related studies. We have developed a novel system of full-field optical coherence tomography (FF-OCT) for noninvasive 3D subcellular live imaging of preimplantation mouse embryos with no need of dye labeling. 3D digitized embryos can be obtained by image processing. Label-free 3D live imaging is demonstrated for the mouse embryos at various typical preimplantation stages with a spatial resolution of 0.7 [micro sign]m and imaging rate of 24 fps. Factors that relate to early patterning and polarity, such as pronuclei in zygote, shapes of zona pellucida, location of second polar body, cleavage planes, and the blastocyst axis, can be quantitatively measured. The angle between the two second cleavage planes is accurately measured to be 87 deg. It is shown that FF-OCT provides a potential breakthrough for early patterning, polarity formation, and many other preimplantation-related studies in mammalian developmental biology.

Imaging of oocyte development using ultrahigh-resolution full-field optical coherence tomography

In this paper, a white-light full-field optical coherence tomography is developed to provide three-dimensional imaging of the development of a mouse embryo with ultrahigh-resolution. Spatial resolution of 1.8 μm×1.12 μm (transverse×axial) is achieved owing to the extremely short coherence length of the light source and optimized compensation of dispersion mismatch. A shot-noise limited detection sensitivity of 80 dB is obtained at an acquisition time of 5 seconds per image. To enable in vivo imaging of the mouse embryo development, a homemade incubator is applied to provide appropriate CO2 concentration, temperature, and humidity. An electronic light shutter is used to control the light source in order to avoid unnecessary exposure to the embryo development when the sample is not being scanned. To demonstrate our method, in vivo time series two-dimensional images of the in vitro fertilization process of mouse oocytes at the germinal vesicles stage are presented.

Aperture effect correction in spectroscopic full-field optical coherence tomography

Spatially resolved spectroscopic optical coherence tomography (OCT) has been demonstrated to be a convenient tool for spectral analysis in turbid media. For a full-field OCT configuration using a Mirau objective in the visible range, we found that the effective numerical aperture varies over the field of view, leading to field-dependent spectral shifts in the reconstructed spectra. Interferograms recorded with quasi-monochromatic lights are theoretically fitted with a general Mirau interference formula, and we propose a numerical correction method for white-light spectroscopy. The method is then tested successfully for the measure of the reflectivity of a plane gold sample.

Spectroscopic polarization-sensitive full-field optical coherence tomography

Full-field optical coherence tomography (FF-OCT) is a recent optical imaging technology based on low-coherence interference microscopy for imaging of semi-transparent samples with ~1 µm spatial resolution. FF-OCT produces en-face tomographic images obtained by arithmetic combination of interferometric images acquired by an array camera. In this paper, we demonstrate a unique multimodal FF-OCT system, capable of measuring simultaneously the intensity, the power spectrum and the phase-retardation of light backscattered by the sample being imaged. Compared to conventional FF-OCT, this multimodal system provides enhanced imaging contrasts at the price of a moderate increase in experimental complexity and cost.

Full-Field and Single-Shot Full-Field Optical Coherence Tomography: A Novel Technique for Biomedical Imaging Applications

Since its introduction, optical coherence tomography (OCT) technology has advanced dramatically in various field of both clinical and fundamental research. Full-field and Single-shot full-field OCT (FF-OCT and SS-FF-OCT) are alternative OCT concepts, which aims to improve the image acquisition speed and to simplify the optical setup of conventional point-scan OCT by realizing direct line field or full-field sample imaging onto an array or line detector such as CCD or CMOS camera. FF-OCT and SS-FF-OCT are based on bulk optics Linnik-type Michelson interferometer with relatively high numerical aperture (NA) microscopic objectives. This paper will give you an overview of the principle of various types of FF-OCT and SS-FF-OCT techniques and its associated system design concept and image reconstruction algorithms.

Full-field optical coherence tomography using nematic liquid-crystal phase shifter

We present a polarization Linnik interference microscope with a nematic liquid-crystal (NLC) phase shifter for full-field optical coherence tomography of high-quality images. The rotating half-wave plate in conventional achromatic phase shifters was replaced by three liquid-crystal (LC) half-wave plates for implementing three-step phase-shifting interferometry. Thus, the NLC device generates phase shifts quickly and has no vibrations. In addition, the phase shift can be set to an arbitrary value between 0 and 2π by altering the azimuth angles of the LC cells. A tomographic image is retrieved from three sequential phase-shifted interferograms by using a three-step algorithm. The experimental results confirm the feasibility of the proposed technology.

Ultrahigh-resolution full-field optical coherence tomography using spatial coherence gating and quasi-monochromatic illumination

We developed an ultrahigh-resolution full-field optical coherence tomography (FF-OCT) microscope that is based on the spatial, rather than the temporal, coherence gating. The microscope is capable of observing three-dimensional microbiological structures as small as 0.4 μm × 0.4 μm × 1.0 μm (xyz) using quasi-monochromatic light and a liquid crystal retarder. Unlike traditional FF-OCT systems, this microscope can be operated in high resolution for any preferable wavelength with minimized defocusing and dispersion effects. High-resolution images of an onion cell are presented.

Numerical correction of distorted images in full-field optical coherence tomography

We propose a numerical method which can numerically correct the distorted en face images obtained with a full field optical coherence tomography (FF-OCT) system. It is shown that the FF-OCT image of the deep region of a biological sample is easily blurred or degraded because the sample has a refractive index (RI) much higher than its surrounding medium in general. It is analyzed that the focal plane of the imaging system is segregated from the imaging plane of the coherence-gated system due to the RI mismatch. This image-blurring phenomenon is experimentally confirmed by imaging the chrome pattern of a resolution test target through its glass substrate in water. Moreover, we demonstrate that the blurred image can be appreciably corrected by using the numerical correction process based on the Fresnel–Kirchhoff diffraction theory. The proposed correction method is applied to enhance the image of a human hair, which permits the distinct identification of the melanin granules inside the cortex layer of the hair shaft.