Back Cover: Prolonged in vivo functional assessment of the mouse oviduct using optical coherence tomography through a dorsal imaging window (J. Biophotonics 5/2018)

Optical coherence tomography through an implanted dorsal imaging window allows for prolonged in vivo structural and functional assessment of the mouse oviduct (Fallopian tube), including threedimensional structural imaging, quantitative measurements of the smooth muscle contraction, and mapping of cilia beat frequency. This method brings new opportunities for live studies and longitudinal analyses of mouse reproductive events in the native context. Further details can be found in the article by Shang Wang et al. ( e201700316 ).

     

Staging mouse preimplantation development in vivo using optical coherence microscopy

In mammals, preimplantation development primarily occurs in the oviduct (or fallopian tube) where fertilized oocytes migrate through, develop and divide as they prepare for implantation in the uterus. Studies of preimplantation development currently rely on ex vivo experiments with the embryos cultured outside of the oviduct, neglecting the native environment for embryonic growth. This prevents the understanding of the natural process of preimplantation development and the roles of the oviduct in early embryonic health. Here, we report an in vivo optical imaging approach enabling high‐resolution visualizations of developing embryos in the mouse oviduct. By combining optical coherence microscopy (OCM) and a dorsal imaging window, the subcellular structures and morphologies of unfertilized oocytes, zygotes and preimplantation embryos can be well resolved in vivo, allowing for the staging of development. We present the results together with bright‐field microscopy images to show the comparable imaging quality. As the mouse is a well‐established model with a variety of genetic engineering strategies available, the in vivo imaging approach opens great opportunities to investigate how the oviduct and early embryos interact to prepare for successful implantation. This knowledge could have beneficial impact on understanding infertility and improving in vitro fertilization. OCM through a dorsal imaging window enables high‐resolution imaging and staging of mouse preimplantation embryos in vivo in the oviduct.      

Optical coherence tomography for dynamic investigation of mammalian reproductive processes

The biological events associated with mammalian reproductive processes are highly dynamic and tightly regulated by molecular, genetic, and biomechanical factors. Implementation of live imaging in reproductive research is vital for the advancement of our understanding of normal reproductive physiology and for improving the management of reproductive disorders. Optical coherence tomography (OCT) is emerging as a promising tool for dynamic volumetric imaging of various reproductive processes in mice and other animal models. In this review, we summarize recent studies employing OCT-based approaches toward the investigation of reproductive processes in both, males and females. We describe how OCT can be applied to study structural features of the male reproductive system and sperm transport through the male reproductive tract. We review OCT applications for in vitro and dynamic in vivo imaging of the female reproductive system, staging and tracking of oocytes and embryos, and investigations of the oocyte/embryo transport through the oviduct. We describe how the functional OCT approach can be applied to the analysis of cilia dynamics within the male and female reproductive systems. We also discuss the areas of research, where OCT could find potential applications to progress our understanding of normal reproductive physiology and reproductive disorders.     

Real‐time in vivo imaging of adult Zebrafish brain using optical coherence tomography

We report noninvasive imaging of the brain of adult Zebrafish (Danio rerio) using real time optical coherence tomography (OCT) capable of acquiring cross sectional 2D OCT images @ 8 frames/sec. Anatomic features such as telencephalon, tectum opticum, eminentia Granularis and cerebellum were clearly resolved in the OCT images. A 3D model of Zebrafish brain was reconstructed, for the first time to our knowledge, using these 2D OCT images. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

In vivo monitoring blood‐brain barrier permeability using spectral imaging through optical clearing skull window

The blood‐brain barrier (BBB) plays a key role in the health of the central nervous system. Opening the BBB is very important for drug delivery to brain tissues to enhance the therapeutic effect on brain diseases. It is necessary to in vivo monitor the BBB permeability for assessing drug release with high resolution; however, an effective method is lacking. In this work, we developed a new method that combined spectral imaging with an optical clearing skull window to in vivo dynamically monitor BBB opening caused by 5‐aminolevulinic acid (5‐ALA)‐mediated photodynamic therapy (PDT), in which the Evans blue dye (EBd) acted as an indicator of the BBB permeability. Using this method, we effectively monitored the cerebrovascular EBd leakage process. Moreover, the analysis of changes in the vascular and extravascular EBd concentrations demonstrated that the PDT‐induced BBB opening exhibited spatiotemporal differences in the cortex. This spectral imaging method based on the optical clearing skull window provides a low‐cost and simply operated tool for in vivo monitoring BBB opening process. This has a high potential for the visualization of drug delivery to the central nervous system. Thus, it is of tremendous significance in brain disease therapy. Monitoring the changes in PDT‐induced BBB permeability by evaluating the EBd concentration using an optical clearing skull window. (A) Entire brains and coronal sections following treatment of PDT with/without an optical clearing skull window after injection of EBd. (B) Typical EBd distribution maps before and after laser irradiation captured by the spectral imaging method. (Colorbar represents the EBd concentration).      

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.     

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)     

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.     

Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart

Efficient separation of blood and cardiac wall in the beating embryonic heart is essential and critical for experiment‐based computational modelling and analysis of early‐stage cardiac biomechanics. Although speckle variance optical coherence tomography (SV‐OCT) relying on calculation of intensity variance over consecutively acquired frames is a powerful approach for segmentation of fluid flow from static tissue, application of this method in the beating embryonic heart remains challenging because moving structures generate SV signal indistinguishable from the blood. Here, we demonstrate a modified four‐dimensional SV‐OCT approach that effectively separates the blood flow from the dynamic heart wall in the beating mouse embryonic heart. The method takes advantage of the periodic motion of the cardiac wall and is based on calculation of the SV signal over the frames corresponding to the same phase of the heartbeat cycle. Through comparison with Doppler OCT imaging, we validate this speckle‐based approach and show advantages in its insensitiveness to the flow direction and velocity as well as reduced influence from the heart wall movement. This approach has a potential in variety of applications relying on visualization and segmentation of blood flow in periodically moving structures, such as mechanical simulation studies and finite element modelling. Picture : Four‐dimensional speckle variance OCT imaging shows the blood flow inside the beating heart of an E8.5 mouse embryo.     

High‐resolution deep functional imaging of the whole mouse brain by photoacoustic computed tomography in vivo

Photoacoustic computed tomography (PACT) is a non‐invasive imaging technique offering high contrast, high resolution, and deep penetration in biological tissues. We report a PACT system equipped with a high frequency linear transducer array for mapping the microvascular network of a whole mouse brain with the skull intact and studying its hemodynamic activities. The linear array was scanned in the coronal plane to collect data from different angles, and full‐view images were synthesized from the limited‐view images in which vessels were only partially revealed. We investigated spontaneous neural activities in the deep brain by monitoring the concentration of hemoglobin in the blood vessels and observed strong interhemispherical correlations between several chosen functional regions, both in the cortical layer and in the deep regions. We also studied neural activities during an epileptic seizure and observed the epileptic wave spreading around the injection site and the wave propagating in the opposite hemisphere.

     

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)     

Pilot feasibility study of in vivo intraoperative quantitative optical coherence tomography of human brain tissue during glioma resection

This study investigates the feasibility of in vivo quantitative optical coherence tomography (OCT) of human brain tissue during glioma resection surgery in six patients. High‐resolution detection of glioma tissue may allow precise and thorough tumor resection while preserving functional brain areas, and improving overall survival. In this study, in vivo 3D OCT datasets were collected during standard surgical procedure, before and after partial resection of the tumor, both from glioma tissue and normal parenchyma. Subsequently, the attenuation coefficient was extracted from the OCT datasets using an automated and validated algorithm. The cortical measurements yield a mean attenuation coefficient of 3.8 ± 1.2 mm^?1 for normal brain tissue and 3.6 ± 1.1 mm^?1 for glioma tissue. The subcortical measurements yield a mean attenuation coefficient of 5.7 ± 2.1 and 4.5 ± 1.6 mm^?1 for, respectively, normal brain tissue and glioma. Although the results are inconclusive with respect to trends in attenuation coefficient between normal and glioma tissue due to the small sample size, the results are in the range of previously reported values. Therefore, we conclude that the proposed method for quantitative in vivo OCT of human brain tissue is feasible during glioma resection surgery.     

Hyperspectral optical coherence tomography for in vivo visualization of melanin in the retinal pigment epithelium

Previous studies for melanin visualization in the retinal pigment epithelium (RPE) have exploited either its absorption properties (using photoacoustic tomography or photothermal optical coherence tomography [OCT]) or its depolarization properties (using polarization sensitive OCT). However, these methods are only suitable when the melanin concentration is sufficiently high. In this work, we present the concept of hyperspectral OCT for melanin visualization in the RPE when the concentration is low. Based on white light OCT, a hyperspectral stack of 27 wavelengths (440‐700 nm) was created in post‐processing for each depth‐resolved image. Owing to the size and shape of the melanin granules in the RPE, the variations in backscattering coefficient as a function of wavelength could be identified—a result which is to be expected from Mie theory. This effect was successfully identified both in eumelanin‐containing phantoms and in vivo in the low‐concentration Brown Norway rat RPE.      

In vivo noninvasive measurement of spatially resolved corneal elasticity in human eyes using Lamb wave optical coherence elastography

Current elastography techniques are limited in application to accurately assess spatially resolved corneal elasticity in vivo for human eyes. The air‐puff optical coherence elastography (OCE) with an eye motion artifacts correction algorithm is developed to distinguish the in vivo cornea vibration from the eye motion and visualize the Lamb wave propagation clearly in healthy subjects. Based on the Lamb wave model, the phase velocity dispersion curve in the high‐frequency is calculated to obtain spatially resolved corneal elasticity accurately with high repeatability. It is found that the corneal elasticity has regional variations and is correlated with intraocular pressure, which suggests that the method has the potential to provide noninvasive measurement of spatially resolved corneal elasticity in clinical practice.     

Optical coherence tomography as a noninvasive 3D real time imaging tool for the rapid evaluation of phenotypic variations in insect embryonic development

Noninvasive visualization of embryos at different development stages is crucial for the understanding of the basic developmental biology. It is therefore desirable to have an imaging tool capable of rapidly evaluating the effects of gene manipulation or genome editing in developing embryos for the studies of gene functions and genetic engineering. Here, we propose and demonstrate a novel use of optical coherence tomography (OCT) to noninvasively exam the embryonic development of the migratory locusts in real time with 3‐dimensional (3D) view capability. In particular, we obtain the sufficiently high spatial resolution tomographic 2D and 3D images of live locust embryos throughout their development processes. We show that not only we are able to noninvasively observe all previously known forms of blastokinesis as an embryo develops, such as anatrepsis, katatrepsis, revolution, rotation and diapauses, and determine their precise occurring time or duration, but also discover an unreported rotation form we named “twist.” In addition, with the OCT images we determined the exact occurring time of diapauses of the locusts from Tibetan plateau for the first time. Finally, we demonstrate that OCT systems can be used to rapidly capture the development defects of genetically modified embryos in which certain genes essential for embryonic development were suppressed by RNA interference. Our work shows that OCT is an enabling imaging tool with sufficient spatial resolution for the rapid evaluation of embryonic variations of small animals.     

Automated three‐dimensional cell counting method for grading uveitis of rodent eye in vivo with optical coherence tomography

In preclinical vision research, cell grading in small animal models is essential for the quantitative evaluation of intraocular inflammation. Here, we present a new and practical optical coherence tomography (OCT) image analysis method for the automated detection and counting of aqueous cells in the anterior chamber (AC) of a rodent model of uveitis. Anterior segment OCT images are acquired with a 100 kHz swept‐source OCT system. The proposed method consists of 2 steps. In the first step, we first despeckle and binarize each OCT image. After removing AS structures in the binary image, we then apply area thresholding to isolate cell‐like objects. Potential cell candidates are selected based on their best fit to roundness. The second step performs the cell counting within the whole AC, in which additional cell tracking analysis is conducted on the successive OCT images to eliminate redundancy in cell counting. Finally, 3D cell grading using the proposed method is demonstrated in longitudinal OCT imaging of a mouse model of anterior uveitis in vivo. Rendering of anterior segment (orange) of mouse eye and automatically counted anterior chamber cells (green). Inset is a top view of the rendering, showing the cell distribution across the anterior chamber.      

In vivo detection of endotracheal tube biofilms in intubated critical care patients using catheter‐based optical coherence tomography

The formation of biofilms in the endotracheal tubes (ETTs) of intubated patients on mechanical ventilation is associated with a greater risk of ventilator‐associated pneumonia and death. New technologies are needed to detect and monitor ETTs in vivo for the presence of these biofilms. Longitudinal OCT imaging was performed in mechanically ventilated subjects at 24‐hour intervals until extubation to detect the formation and temporal changes of in vivo ETT biofilms. OCT‐derived attenuation coefficient images were used to differentiate between mucus and biofilm. Extubated ETTs were examined with optical and electron microscopy, and all imaging results were correlated with standard‐of‐care clinical test reports. OCT and attenuation coefficient images from four subjects were positive for ETT biofilms and were negative for two subjects. The processed and stained extubated ETTs and clinical reports confirmed the presence/absence of biofilms in all subjects. Our findings confirm that OCT can detect and differentiate between biofilm‐positive and biofilm‐negative groups (P < 10^?5). OCT image‐based features may serve as biomarkers for direct in vivo detection of ETT biofilms and help drive investigation of new management strategies to reduce the incidence of VAP.      

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.     

Assessment of the influence of viscoelasticity of cornea in animal ex vivo model using air‐puff optical coherence tomography and corneal hysteresis

Application of the air‐puff swept source optical coherence tomography (SS‐OCT) instrument to determine the influence of viscoelasticity on the relation between overall the air‐puff force and corneal apex displacement of porcine corneas ex vivo is demonstrated. Simultaneous recording of time‐evolution of the tissue displacement and air pulse stimulus allows obtaining valuable information related in part to the mechanical properties of the cornea. A novel approach based on quantitative analysis of the corneal hysteresis of OCT data is presented. The corneal response to the air pulse is assessed for different well‐controlled intraocular pressure (IOP) levels and for the progression of cross‐linking‐induced stiffness of the cornea. Micrometer resolution, fast acquisition and noncontact character of the air‐puff SS‐OCT measurements have potential to improve the in vivo assessment of mechanical properties of the human corneas.      

Long‐term and in vivo assessment of Aβ protein‐induced brain atrophy in a zebrafish model by optical coherence tomography

In this study, a neurotoxicity model of zebrafish induced by amyloid beta (Aβ) protein was developed and evaluated in vivo by optical coherence tomography (OCT). Aβ protein and phosphate buffer saline (PBS) were separately injected into the head of two groups of adult zebrafish (n = 6 per group). Congo‐red staining results confirmed that Aβ protein had penetrated into brain tissue. All zebrafish were imaged with OCT on the 0th, 5th, 10th, 15th and 20th day postinjection. OCT images showed that PBS is not toxic to brain tissue. However, significant brain atrophy could be seen in the OCT images of zebrafish injected with Aβ‐protein that was verified by histological consequences. In addition, zebrafish in the model group showed memory decline in behavioral tests. This study verified the feasibility of in vivo long‐term assessment of Aβ protein‐induced brain atrophy in adult zebrafish by OCT that has great potential to be applied in the neurological diseases research.