In vivo pump‐probe optical coherence tomography imaging in Xenopus laevis

Currently, optical coherence tomography (OCT), is not capable of obtaining molecular information often crucial for identification of disease. To enable molecular imaging with OCT, we have further developed a technique that harnesses transient changes in light absorption in the sample to garner molecular information. A Fourier‐domain Pump‐Probe OCT (PPOCT) system utilizing a 532 nm pump and 830 nm probe has been developed for imaging hemoglobin. Methylene blue, a biological dye with well‐know photophysics, was used to characterize the system before investigating the origin of the hemoglobin PPOCT signal. The first in vivo PPOCT images were recorded of the vasculature in Xenopus laevis. The technique was shown to work equally well in flowing and nonflowing vessels. Furthermore, PPOCT was compared with other OCT extensions which require flow, such as Doppler OCT and phase‐variance OCT. PPOCT was shown to better delineate tortuous vessels, where nodes often restrict Doppler and phase‐variance reconstruction. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Four‐dimensional imaging of murine subpleural alveoli using high‐speed optical coherence tomography

The investigation of lung dynamics on alveolar scale is crucial for the understanding and treatment of lung diseases, such as acute lung injury and ventilator induced lung injury, and to promote the development of protective ventilation strategies. One approach to this is the establishment of numerical simulations of lung tissue mechanics where detailed knowledge about three‐dimensional alveolar structure changes during the ventilation cycle is required. We suggest four‐dimensional optical coherence tomography (OCT) imaging as a promising modality for visualizing the structural dynamics of single alveoli in subpleural lung tissue with high temporal resolution using a mouse model. A high‐speed OCT setup based on Fourier domain mode locked laser technology facilitated the acquisition of alveolar structures without noticeable motion artifacts at a rate of 17 three‐dimensional stacks per ventilation cycle. The four‐dimensional information, acquired in one single ventilation cycle, allowed calculating the volume‐pressure curve and the alveolar compliance for single alveoli. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Prolonged in vivo functional assessment of the mouse oviduct using optical coherence tomography through a dorsal imaging window

The oviduct (or fallopian tube) serves as an environment for gamete transport, fertilization and preimplantation embryo development in mammals. Although there has been increasing evidence linking infertility with disrupted oviduct function, the specific roles that the oviduct plays in both normal and impaired reproductive processes remain unclear. The mouse is an important mammalian model to study human reproduction. However, most of the current analyses of the mouse oviduct rely on static histology or 2D visualization, and are unable to provide dynamic and volumetric characterization of this organ. The lack of imaging access prevents longitudinal live analysis of the oviduct and its associated reproductive events, limiting the understanding of mechanistic aspects of fertilization and preimplantation pregnancy. To address this limitation, we report a 3D imaging approach that enables prolonged functional assessment of the mouse oviduct in vivo. By combining optical coherence tomography with a dorsal imaging window, this method allows for extended volumetric visualization of the oviduct dynamics, which was previously not achievable. The approach is used for quantitative analysis of oviduct contraction, spatiotemporal characterization of cilia beat frequency and longitudinal imaging. This new approach is a useful in vivo imaging platform for a variety of live studies in mammalian reproduction.      

Comparative study of optical coherence tomography angiography algorithms for rodent retinal imaging

Optical coherence tomography angiography (OCTA) is a functional extension of optical coherence tomography for non-invasive in vivo three-dimensional imaging of the microvasculature of biological tissues. Several algorithms have been developed to construct OCTA images from the measured optical coherence tomography signals. In this study, we compared the performance of three OCTA algorithms that are based on the variance of phase, amplitude, and the complex representations of the optical coherence tomography signals for rodent retinal imaging, namely the phase variance, improved speckle contrast, and optical microangiography. The performance of the different algorithms was evaluated by comparing the quality of the OCTA images regarding how well the vasculature network can be resolved. Quantities that are widely used in ophthalmic studies including blood vessel density, vessel diameter index, vessel perimeter index, vessel complexity index were also compared. Results showed that both the improved speckle contrast and optical microangiography algorithms are more robust than phase variance, and they can reveal similar vasculature features while there are statistical differences in the calculated quantities.     

Multicontrast endomyocardial imaging by single‐channel high‐resolution cross‐polarization optical coherence tomography

A single‐channel high‐resolution cross‐polarization (CP) optical coherence tomography (OCT) system is presented for multicontrast imaging of human myocardium in one‐shot measurement. The intensity and functional contrasts, including the ratio between the cross‐ and co‐polarization channels as well as the cumulative retardation, are reconstructed from the CP‐OCT readout. By comparing the CP‐OCT results with histological analysis, it is shown that the system can successfully delineate microstructures in the myocardium and differentiate the fibrotic myocardium from normal or ablated myocardium based on the functional contrasts provided by the CP‐OCT system. The feasibility of using A‐line profiles from the 2 orthogonal polarization channels to identify fibrotic myocardium, normal myocardium and ablated lesion is also discussed.      

Flexible wide‐field optical micro‐angiography based on Fourier‐domain multiplexed dual‐beam swept source optical coherence tomography

Wide‐field optical coherence tomography angiography (OCTA) is gaining interest in clinical imaging applications. In this pursuit, it is challenging to maintain the imaging resolution and sensitivity throughout the wide field of view (FoV). Here, we propose a novel method/system of dual‐beam arrangement and Fourier‐domain multiplexing to achieve wide‐field OCTA when imaging the uneven surface samples. The proposed system provides 2 separate FoVs, with flexibility that the imaging area, focus of the imaging beam and imaging depth range can be individually adjusted for each FoV, leading to either (1) increased system imaging FoV or (2) capability of targeting 2 regions of interests that locate at depths with large difference between each other. We demonstrate this novel method by employing 100 kHz laser source in a swept source OCTA to achieve an effective 200 kHz sweeping rate, covering a 22 × 22 mm FoV. The results are verified by a SS‐OCTA system employing a 200 kHz laser source, together with the experimental demonstrations when imaging whole brain vasculature in rodent models and skin blood perfusion in human fingers, show‐casing the capability of proposed system to image live large samples with complex surface topography.      

Three-dimensional segmentation and depth-encoded visualization of choroidal vasculature using swept-source optical coherence tomography

The choroid provides nutritional support for the retinal pigment epithelium and photoreceptors. Choroidal dysfunction plays a major role in several of the most important causes of vision loss including age-related macular degeneration, myopic degeneration, and pachychoroid diseases such as central serous chorioretinopathy and polypoidal choroidal vasculopathy. We describe an imaging technique using depth-resolved swept-source optical coherence tomography (SS-OCT) that provides full-thickness three-dimensional (3D) visualization of choroidal anatomy including topographical features of individual vessels. Enrolled subjects with different clinical manifestations within the pachychoroid disease spectrum underwent 15 mm × 9 mm volume scans centered on the fovea. A fully automated method segmented the choroidal vessels using their hyporeflective lumens. Binarized choroidal vessels were rendered in a 3D viewer as a vascular network within a choroidal slab. The network of choroidal vessels was color depth-encoded with a reference to the Bruch’s membrane segmentation. Topographical features of the choroidal vasculature were characterized and compared with choroidal imaging obtained with indocyanine green angiography (ICGA) from the same subject. The en face SS-OCT projections of the larger choroid vessels closely resembled to that obtained with ICGA, with the automated SS-OCT approach proving additional depth-encoded 3D information. In 16 eyes with pachychoroid disease, the SS-OCT approach added clinically relevant structural details, including choroidal thickness and vessel depth, which the ICGA studies could not provide. Our technique appears to advance the in vivo visualization of the full-thickness choroid, successfully reveals the topographical features of choroidal vasculature, and shows potential for further quantitative analysis when compared with other choroidal imaging techniques. This improved visualization of choroidal vasculature and its 3D structure should provide an insight into choroid-related disease mechanisms as well as their responses to treatment.     

Imaging growth dynamics of tumour spheroids using optical coherence tomography

Optical coherence tomography (OCT) was used to monitor the dynamics of tumour spheroid formation by the hanging drop method. In contrast to microscopy, the estimates obtained using OCT for the volume of the spheroid, were consistent with the measured changes in cell number as a function of time. The OCT images also revealed heterogeneous structures in the spheroids of ∼200 μm diameter. These corresponded to the necrotic regions identified by fluorescence of propidium iodide stained cells.     

Measuring polarization changes in the human outer retina with polarization‐sensitive optical coherence tomography

Morphological changes in the outer retina such as drusen are established biomarkers to diagnose age‐related macular degeneration. However, earlier diagnosis might be possible by taking advantage of more subtle changes that accompany tissues that bear polarization‐altering properties. To test this hypothesis, we developed a method based on polarization‐sensitive optical coherence tomography with which volumetric data sets of the macula were obtained from 10 young (<25 years) and 10 older (>54 years) subjects. All young subjects and 5 of the older subjects had retardance values induced by the retinal pigment epithelium and Bruch's membrane (RPE‐BM) complex that were just above the noise floor measurement (5°‐13° at 840 nm). In contrast, elevated retardance, up to 180°, was observed in the other 5 older subjects. Analysis of the degree of polarization uniformity (DOPU) demonstrates that reduced DOPU (<0.4) in the RPE is associated with elevated double pass phase retardation (DPPR) below the RPE‐BM complex, suggesting that the observed elevated DPPR in older subjects is the result of increased scattering or polarization scrambling. Collectively, our measurements show that the outer retina can undergo dramatic change in its polarization properties with age, and in some cases still retain its clinically normal appearance.      

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.      

Super‐achromatic optical coherence tomography capsule for ultrahigh‐resolution imaging of esophagus

Endoscopic optical coherence tomography (OCT) is a noninvasive technology allowing for imaging of tissue microanatomies of luminal organs in real time. Conventional endoscopic OCT operates at 1300 nm wavelength region with a suboptimal axial resolution limited to 8‐20 μm. In this paper, we present the first ultrahigh‐resolution tethered OCT capsule operating at 800 nm and offering about 3‐ to 4‐fold improvement of axial resolution (plus enhanced imaging contrast). The capsule uses diffractive optics to manage chromatic aberration over a full ~200 nm spectral bandwidth centering around 830 nm, enabling to achieve super‐achromaticity and an axial resolution of ~2.6 μm in air. The performance of the OCT capsule is demonstrated by volumetric imaging of swine esophagus ex vivo and sheep esophagus in vivo, where fine anatomic structures including the sub‐epithelial layers are clearly identified. The ultrahigh resolution and excellent imaging contrast at 800 nm of the tethered capsule suggest the potential of the technology as an enabling tool for surveillance of early esophageal diseases on awake patients without the need for sedation.      

Three‐dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

A preliminary clinical trial using state‐of‐the‐art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three‐dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub‐cellular details with axial and transverse resolutions of <2 μm and <0.5 μm, respectively, from a volume of 0.35 × 0.35 × 0.2 mm³ at a frame rate of 0.14 Hz (512 × 512 pixels). State‐of‐the‐art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions ～8 μm and ～20 μm, respectively, from a volume of 7 × 3.5 × 1.5 mm³ at a frame rate of 46 Hz (1024 × 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre‐screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub‐cellular level information of the respective regions (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High‐definition optical coherence tomography of melanocytic skin lesions

High‐definition optical coherence tomography (HD‐OCT) scanners have recently been developed. We assessed micromorphological HD‐OCT correlates of benign naevi (BN) and malignant melanoma (MM). 28 BN and 20 MM were studied using HD‐OCT and histology. Epidermal honeycomb/cobblestone pattern, regular junctional cell nests, and edged papillae are more often observed in BN, whereas fusion of rete ridges, pagetoid cells and junctional and/or dermal nests with atypical cells are more frequently seen in MM. A high overlap of HD‐OCT features in BN and MM was observed and in 20% of MM we did not find evidence for malignancy in OCT images at all. Using HD‐OCT it is possible to visualize architectural and cellular alterations of melanocytic skin lesions. The overlap of HD‐OCT features seen in BN and MM and the absence of suspicious HD‐OCT features in some MM represents an important limitation of HD‐OCT affecting the sensitivity of HD‐OCT in diagnosing MM.

High‐definition optical coherence tomography and the corresponding vertically sectioned histology of a compound naevus.     

Cross‐scanning optical coherence tomography angiography for eye motion correction

We propose a cross‐scanning optical coherence tomography (CS‐OCT) system to correct eye motion artifacts in OCT angiography images. This system employs a dual‐illumination configuration with two orthogonally polarized beams, each of which simultaneously perform raster scanning in perpendicular direction with each other over the same area. In the reference arm, a polarization delay unit is used to acquire the two orthogonally polarized interferograms with a single photo detector by introducing different optical delay lines. The two cross‐scanned volume data are affected by the same eye motion but in two orthogonal directions. We developed a motion correction algorithm, which removes artifacts in the slow axis of each angiogram using the other and merges them through a nonrigid registration algorithm. In this manner, we obtained a motion‐corrected angiogram within a single volume scanning time without additional eye‐tracking devices.     

Retinal imaging with optical coherence tomography and low‐loss adaptive optics using a 2.8‐mm beam size

As data acquisition for retinal imaging with optical coherence tomography (OCT) becomes faster, efficient collection of photons becomes more important to maintain image quality. One approach is to use a larger aperture at the eye's pupil to collect more photons that have been reflected from the retina. A 2.8‐mm beam diameter system with only seven reflecting surfaces was developed for low‐loss retinal imaging. The larger beam size requires defocus and astigmatism correction, which was done in a closed loop adaptive optics method using a Shack‐Hartmann wavefront sensor and a deformable mirror (DM) with 140 actuators and a ±2.75 μm stroke. This DM facilitates defocus correction ranging from approximately ?3 D to +3 D. Comparing the new system with a standard 1.2‐mm system on a model eye, a signal‐to‐noise gain of 4.5 dB and a 2.3 times smaller speckle size were measured. Measurements on the retinas of five subjects showed even better results, with increases in dynamic range up to 13 dB. Note that the new sample arm only occupies 30 cm × 60 cm, which makes it highly suitable for imaging in a clinical environment. Figure: B‐scan images obtained over a width of 8 deg from the right eye of a 31‐year‐old Caucasian male. While the left side was imaged with a standard 1.2‐mm OCT system, the right side was imaged with the 2.8‐mm system. Both images were collected with the same integration time and incident power, after correction of aberrations. Using the dynamic range within the images, which is determined by comparing the highest pixel value to the noise floor, a difference in dynamic range of 10.8 dB was measured between the two systems.      

In vivo detection of tumor boundary using ultrahigh‐resolution optical coherence angiography and fluorescence imaging

Accurate detection of early tumor margin is of great preclinical and clinical implications for predicting the survival rate of subjects and assessing the response of tumor microenvironment to chemotherapy or radiation therapy. Here, we report a multimodality optical imaging study on in vivo detection of tumor boundary by analyzing neoangiogenesis of tumor microenvironment (microangiography), microcirculatory blood flow (optical Doppler tomography) and tumor proliferation (green fluorescent protein [GFP] fluorescence). Microangiography demonstrates superior sensitivity (77.7 ± 6.4%) and specificity (98.2 ± 1.7%) over other imaging technologies (eg, optical coherence tomography) for tumor margin detection. Additionally, we report longitudinal in vivo imaging of tumor progression and show that the abrupt tumor cell proliferation did not occur until local capillary density and cerebral blood flow reached their peak approximately 2 weeks after tumor implantation. The unique capability of longitudinal multimodality imaging of tumor angiogenesis may provide new insights in tumor biology and in vivo assessment of the treatment effects on anti‐angiogenesis therapy for brain cancer.     

In vivo observation of metamorphosis of Plodia interpunctella Hübner using three‐dimensional optical coherence tomography

Morphological assessment and three‐dimensional reconstructions of internal structures of Plodia interpunctella Hübner during metamorphosis stages were experimentally demonstrated using optical coherence tomography (OCT) for the first time. The conventional, complex sectioning methods were significantly simplified owing to the non‐invasive three‐dimensional imaging capability of OCT. Further, this study demonstrates the use of OCT as a non‐invasive detection tool for in vivo morphological observation of metamorphosis stages to gain a better understanding about the growth of internal organs, which can be considered a useful discovery in the field of entomology. Thus, the metamorphosis stages starting from the larva, three pupa stages to the adult stage were periodically visualized to examine the development of internal organs at each specific stage. This study essentially offers real‐time morphological information by non‐destructive observation of the organism and can also be useful for the investigation of other agricultural pests.     

Quantification of diabetic macular ischemia using novel three‐dimensional optical coherence tomography angiography metrics

We applied three‐dimensional (3D) analysis to optical coherence tomography angiography (OCTA) to measure macular ischemia in eyes affected by non‐proliferative diabetic retinopathy (DR). A previously validated algorithm was applied to OCTA data in order to obtain 3D visualization of the retinal vasculature. Successively, a global thresholding algorithm was applied and two novel quantitative metrics were introduced: 3D vascular volume and 3D perfusion density. Two‐dimensional (2D) OCTA metrics were also obtained with different binarization thresholds for comparison. Of the 30 patients included, 15 were diagnosed with DR and 15 were controls. The 3D vascular volume and 3D perfusion density were reduced in DR eyes (P < .0001). The 2D variables also significantly differ between groups. The 3D perfusion density had the highest area under the receiver operating characteristic curve (0.964) among tested variables. Assessing quantitative perfusion using 3D analysis is reliable and promising, and with an elevated diagnostic efficacy in identifying DR eyes.     

Origin of improved depth penetration in dual‐axis optical coherence tomography: a Monte Carlo study

Recent studies have demonstrated that extended imaging depth can be achieved using dual‐axis optical coherence tomography (DA‐OCT). By illuminating and collecting at an oblique angle, multiple forward scattered photons from large probing depths are preferentially detected. However, the mechanism behind the enhancement of imaging depth needs further illumination. Here, the signal of a DA‐OCT system is studied using a Monte Carlo simulation. We modeled light transport in tissue and recorded the spatial and angular distribution of photons exiting the tissue surface. Results indicate that the spatial separation and offset angle created by the non‐telecentric scanning configuration promote the collection of more deeply propagating photons than conventional on‐axis OCT.      

K‐distribution three‐dimensional mapping of biological tissues in optical coherence tomography

Probability density function (PDF) analysis with K‐distribution model of optical coherence tomography (OCT) intensity signals has previously yielded a good representation of the average number of scatterers in a coherence volume for microspheres‐in‐water systems, and has shown initial promise for biological tissue characterization. In this work, we extend these previous findings, based on single point M‐mode or two‐dimenstional slice analysis, to full three‐dimensional (3D) imaging maps of the shape parameter α of the K‐distribution PDF. After selecting a suitably sized 3D evaluation window, and verifying methodology in phantoms, the resultant parametric α images obtained in different animal tissues (rat liver and brain) show new contrasting ability not seen in conventional OCT intensity images.