Interplane bulk motion analysis and removal based on normalized cross‐correlation in optical coherence tomography angiography

Bulk motion seriously degrades the image quality of optical coherence tomography angiography (OCTA). Conventional correction methods focus on in‐plane displacement, while the bulk motion component perpendicular to B‐scans also introduces noise. This work first presents an evaluation of this component using a specific scan protocol and an approximate expression derived from peak‐normalized cross‐correlation values, and then quantitatively assesses how interplane bulk motion noise reduce the sensitivity of cross‐sectional angiograms. Finally, we developed a repetitive bulk motion correction method based on the estimated displacements and redundant volume scans. The correction does not require registration and angiogram reconstruction of low flow sensitivity frames, and the results of in vivo mice skin OCTA imaging experiments show that the proposed method can effectively reduce bulk motion noise caused by cardiac and respiratory motion and occasional shaking, and improve OCTA image quality, which has practical significance for clinical OCTA diagnosis and analysis.     

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.      

Inside Cover: Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

A type of compact and cost‐effective light‐sheet imaging device, termed sub‐voxel‐resolving light‐sheet add‐on module (SLAM), is developed to cooperate with conventional 2D epifluorescence microscope, allowing high‐contrast, resolution‐improved 3D imaging of various biological samples at high throughput. Further details can be found in the article by Fang Zhao, Yicong Yang, Yi Li, et al. ( e201960243 ).

     

A deep‐learning‐based approach for noise reduction in high‐speed optical coherence Doppler tomography

Optical coherence Doppler tomography (ODT) increasingly attracts attention because of its unprecedented advantages with respect to high contrast, capillary‐level resolution and flow speed quantification. However, the trade‐off between the signal‐to‐noise ratio of ODT images and A‐scan sampling density significantly slows down the imaging speed, constraining its clinical applications. To accelerate ODT imaging, a deep‐learning‐based approach is proposed to suppress the overwhelming phase noise from low‐sampling density. To handle the issue of limited paired training datasets, a generative adversarial network is performed to implicitly learn the distribution underlying Doppler phase noise and to generate the synthetic data. Then a 3D based convolutional neural network is trained and applied for the image denoising. We demonstrate this approach outperforms traditional denoise methods in noise reduction and image details preservation, enabling high speed ODT imaging with low A‐scan sampling density.     

Visibility of microvessels in Optical Coherence Tomography angiography depends on angular orientation

Optical Coherence Tomography angiography (OCTA) is a widespread tool for depth‐resolved imaging of chorioretinal vasculature with single microvessel resolution. To improve the clinical interpretation of OCTA, the conditions affecting visualization of microvessels must be defined. Here we inject a scattering plasma tracer (Intralipid) during OCTA imaging of the anesthetized rat eye. In the retina, we find that interlaminar (vertical) vessels that connect laminae have one‐fourth to one‐third the OCTA red blood cell to tracer (RBC‐to‐tracer) signal ratio of intralaminar (horizontal) vessels. This finding suggests that the OCTA signal from microvessels depends on angular orientation, making vertically‐oriented vessels more difficult to visualize using intrinsic contrast alone. Clinicians should be aware of this potential artifact when interpreting OCTA.     

Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy

Light‐sheet fluorescence microscopy (LSFM) allows volumetric live imaging at high‐speed and with low photo‐toxicity. Various LSFM modalities are commercially available, but their size and cost limit their access by the research community. A new method, termed sub‐voxel‐resolving (SVR) light‐sheet add‐on microscopy (SLAM), is presented to enable fast, resolution‐enhanced light‐sheet fluorescence imaging from a conventional wide‐field microscope. This method contains two components: a miniature add‐on device to regular wide‐field microscopes, which contains a horizontal laser light‐sheet illumination path to confine fluorophore excitation at the vicinity of the focal plane for optical sectioning; an off‐axis scanning strategy and a SVR algorithm that utilizes sub‐voxel spatial shifts to reconstruct the image volume that results in a twofold increase in resolution. SLAM method has been applied to observe the muscle activity change of crawling C. elegans, the heartbeat of developing zebrafish embryo, and the neural anatomy of cleared mouse brains, at high spatiotemporal resolution. It provides an efficient and cost‐effective solution to convert the vast number of in‐service microscopes for fast 3D live imaging with voxel‐super‐resolved capability.     

High‐speed X‐ray‐induced luminescence computed tomography

X‐ray‐induced luminescence computed tomography (XLCT) is an emerging molecular imaging. Challenges in improving spatial resolution and reducing the scan time in a whole‐body field of view (FOV) still remain for practical in vivo applications. In this study, we present a novel XLCT technique capable of obtaining three‐dimensional (3D) images from a single snapshot. Specifically, a customed two‐planar‐mirror component is integrated into a cone beam XLCT imaging system to obtain multiple optical views of an object simultaneously. Furthermore, a compressive sensing based algorithm is adopted to improve the efficiency of 3D XLCT image reconstruction. Numerical simulations and experiments were conducted to validate the single snapshot X‐ray‐induced luminescence computed tomography (SS‐XLCT). The results show that the 3D distribution of the nanophosphor targets can be visualized much faster than conventional cone beam XLCT imaging method that was used in our comparisons while maintaining comparable spatial resolution as in conventional XLCT imaging. SS‐XLCT has the potential to harness the power of XLCT for rapid whole‐body in vivo molecular imaging of small animals.     

Dual‐ and single‐shot susceptibility ratio measurements with circular polarizations in second‐harmonic generation microscopy

Polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio , with z‐axis parallel and x‐axis perpendicular to the C₆ symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P‐SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of : A dual‐shot configuration where the SHG circular anisotropy generated using incident right‐ and left‐handed circularly‐polarized light is measured; and a single‐shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes‐Mueller polarimetry. The dual‐ and single‐shot circular anisotropy measurements can be used for fast imaging that is independent of the in‐plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.     

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.     

A novel automatic 3D stitching algorithm for optical coherence tomography angiography and its application in dermatology

Optical coherence tomography (OCT) and OCT angiography (OCTA) techniques offer numerous advantages in clinical skin applications but the field of view (FOV) of current commercial systems are relatively limited to cover the entire skin lesion. The typical method to expand the FOV is to apply wide field objective lens. However, lateral resolution is often sacrificed when scanning with these lenses. To overcome this drawback, we developed an automated 3D stitching method for creating high-resolution skin structure and vascular volumes with large field of view, which was realized by montaging multiple adjacent OCT and OCTA volumes. The proposed stitching method is demonstrated by montaging 3 × 3 OCT and OCTA volumes (nine OCT/OCTA volumes as one data set with each volume covers 2.5 cm × 2.5 cm area) of healthy thin and thick skin from six volunteers. The proposed stitching protocol achieves high flexibility and repeatable for all the participants. Moreover, according to evaluation of structural similarity index and feature similarity index, our proposed stitched result has a superior similarity to single scanning protocol in large-scaled. We had also verified its improved performance through assessing metrics of vessel contrast-noise-ratio (CNR) from 2.07 ± 0.44 (single large-scaled scanning protocol) to 3.05 ± 0.51 (proposed 3 × 3 sub-volume stitching method).     

Hyperspectral characterization of re‐epithelialization in an in vitro wound model

In vitro wound models are useful for research on wound re‐epithelialization. Hyperspectral imaging represents a non‐destructive alternative to histology analysis for detection of re‐epithelialization. This study aims to characterize the main optical behavior of a wound model in order to enable development of detection algorithms. K‐Means clustering and agglomerative analysis were used to group spatial regions based on the spectral behavior, and an inverse photon transport model was used to explain differences in optical properties. Six samples of the wound model were prepared from human tissue and followed over 22 days. Re‐epithelialization occurred at a mean rate of 0.24 mm²/day after day 8 to 10. Suppression of wound spectral features was the main feature characterizing re‐epithelialized and intact tissue. Modeling the photon transport through a diffuse layer placed on top of wound tissue properties reproduced the spectral behavior. The missing top layer represented by wounds is thus optically detectable using hyperspectral imaging.     

Collagen ultrastructural symmetry and its malignant alterations in human breast cancer revealed by polarization‐resolved second‐harmonic generation microscopy

Several specific alterations of the extracellular matrix can be considered a distinctive hallmark of cancer. In particular, a different morphology of the collagen scaffold is frequently found within the peritumoural environment. In this study, we report about a significant difference in the ultrastructural organization of collagen at the supra‐molecular level between the perilesional scaffold and the tumour area in human breast carcinoma samples. In particular, we demonstrated that polarization‐resolved second‐harmonic generation (P‐SHG) microscopy is able to link the altered collagen architecture at the ultrastructural level found in perilesional tissue with a different organization of collagen fibrils at the molecular level.     

Lightsheet fluorescence lifetime imaging microscopy with wide‐field time‐correlated single photon counting

We report on wide‐field time‐correlated single photon counting (TCSPC)‐based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single‐photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide‐field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.     

Optical coherence tomography angiography for mapping cerebral microvasculature based on normalized differentiation analysis

Optical coherence tomography angiography (OCTA) is a label‐free, noninvasive biomedical imaging modality for mapping microvascular networks and quantifying blood flow velocities in vivo. Simple computation and fast processing are critical for the OCTA in some applications. Herein, we report on a normalized differentiation method for mapping cerebral microvasculature with the advantages of simple analysis and high image quality, benefitting from computation of differentiation and characteristics of normalization. Normalized differentiation values are validated to have a nearly linear relationship with flow velocities in a range using a flow phantom. The measurements in a rat cerebral cortex show that the OCTA based on the normalized differentiation analysis can generate microvascular images with high quality and monitor spatiotemporal dynamics of blood flow with simple computation and fast processing before and after localized ischemia induced by arterial occlusion.     

STED nanoscopy of KK114‐stained pathogenic bacteria

Super‐resolution microscopy techniques can provide answers to still pending questions on prokaryotic organisms but are yet to be used at their full potential for this purpose. To address this, we evaluate the ability of the rhodamine‐like KK114 dye to label various types of bacteria, to enable imaging of fine structural details with stimulated emission depletion microscopy (STED). We assessed fluorescent labeling with KK114 for eleven Gram‐positive and Gram‐negative bacterial species and observed that this contrast agent binds to their cell membranes. Significant differences in the labeling outputs were noticed across the tested bacterial species, but importantly, KK114‐staining allowed the observation of subtle nanometric cell details in some cases. For example, a helix pattern resembling a cytoskeleton arrangement was detected in Bacillus subtilis. Furthermore, we found that KK114 easily penetrates the membrane of bacterial microorganism that lost their viability, which can be useful to discriminate between living and dead cells.     

Fan‐shaped scanning approach for miniaturized photoacoustic tomography

We describe a novel scanning approach for miniaturized photoacoustic tomography (PAT), based on fan‐shaped scanning of a single transducer at one or two discrete positions. This approach is tested and evaluated using several phantom and animal experiments. The results obtained show that this new scanning approach provides high image quality in the configuration of miniaturized handheld or endoscopic PAT with improved effective field of view and penetration depth.     

Hyperspectral imaging and characterization of allergic contact dermatitis in the short‐wave infrared

Short‐wave infrared hyperspectral imaging is applied to diagnose and monitor a case of allergic contact dermatitis (ACD) due to poison ivy exposure in one subject. This approach directly demonstrates increased tissue fluid content in ACD lesional skin with a spectral signature that matches the spectral signature of intradermally injected normal saline. The best contrast between the affected and unaffected skin is achieved through a selection of specific wavelengths at 1070, 1340 and 1605 nm and combining them in a pseudo‐red‐green‐blue color space. An image derived from these wavelengths normalized to unaffected skin defines a “tissue fluid index” that may aid in the quantitative diagnosis and monitoring of ACD. Further clinical testing of this promising approach towards disease detection and monitoring with tissue fluid content quantification is warranted.     

In‐vivo hemodynamic imaging of acute prenatal ethanol exposure in fetal brain by photoacoustic tomography

Prenatal ethanol exposure (PEE) can lead to structural and functional abnormalities in fetal brain. Although neural developmental deficits due to PEE have been recognized, the immediate effects of PEE on fetal brain vasculature and hemodynamics remain poorly understood. One of the major obstacles that preclude the rapid advancement of studies on fetal vascular dynamics is the limitation of the imaging techniques. Thus, a technique for noninvasive in‐vivo imaging of fetal vasculature and hemodynamics is desirable. In this study, we explored the dynamic changes of the vessel dimeter, density and oxygen saturation in fetal brain after acute maternal ethanol exposure in the second‐trimester equivalent murine model using a real‐time photoacoustic tomography system we developed for imaging embryo of small animals. The results indicate a significant decrease in fetal brain vessel diameter, perfusion and oxygen saturation. This work demonstrated that PAT can provide high‐resolution noninvasive imaging ability to monitor fetal vascular dynamics.     

Characterization of blood coagulation dynamics and oxygenation in ex‐vivo retinal vessels by fluorescence hyperspectral imaging

Blood coagulation mechanisms forming a blood clot and preventing hemorrhage have been extensively studied in the last decades. Knowing the mechanisms behind becomes very important particularly in the case of blood vessel diseases. Real‐time and accurate diagnostics accompanied by the therapy are particularly needed, for example, in diseases related to retinal vasculature. In our study, we employ for the first time fluorescence hyperspectral imaging (fHSI) combined with the spectral analysis algorithm concept to assess physical as well as functional information of blood coagulation in real‐time. By laser‐induced local disruption of retinal vessels to mimic blood leaking and subsequent coagulation and a proper fitting algorithm, we were able to reveal and quantify the extent of local blood coagulation through direct identification of the change of oxyhemoglobin concentration within few minutes. We confirmed and illuminated the spatio‐temporal evolution of the essential role of erythrocytes in the coagulation cascade as the suppliers of oxygenated hemoglobin. By additional optical tweezers force manipulation, we showed immediate aggregation of erythrocytes at the coagulation site. The presented fluorescence‐based imaging concept could become a valuable tool in various blood coagulation diagnostics as well as theranostic systems if coupled with the laser therapy.     

Label‐free spectroscopic tissue characterization using fluorescence excitation‐scanning spectral imaging

Spectral imaging approaches provide new possibilities for measuring and discriminating fluorescent molecules in living cells and tissues. These approaches often employ tunable filters and robust image processing algorithms to identify many fluorescent labels in a single image set. Here, we present results from a novel spectral imaging technology that scans the fluorescence excitation spectrum, demonstrating that excitation‐scanning hyperspectral image data can discriminate among tissue types and estimate the molecular composition of tissues. This approach allows fast, accurate quantification of many fluorescent species from multivariate image data without the need of exogenous labels or dyes. We evaluated the ability of the excitation‐scanning approach to identify endogenous fluorescence signatures in multiple unlabeled tissue types. Signatures were screened using multi‐pass principal component analysis. Endmember extraction techniques revealed conserved autofluorescent signatures across multiple tissue types. We further examined the ability to detect known molecular signatures by constructing spectral libraries of common endogenous fluorophores and applying multiple spectral analysis techniques on test images from lung, liver and kidney. Spectral deconvolution revealed structure‐specific morphologic contrast generated from pure molecule signatures. These results demonstrate that excitation‐scanning spectral imaging, coupled with spectral imaging processing techniques, provides an approach for discriminating among tissue types and assessing the molecular composition of tissues. Additionally, excitation scanning offers the ability to rapidly screen molecular markers across a range of tissues without using fluorescent labels. This approach lays the groundwork for translation of excitation‐scanning technologies to clinical imaging platforms.