Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second

Non‐invasive biological imaging is crucial for understanding in vivo structure and function. Optical coherence tomography (OCT) and reflectance confocal microscopy are two of the most widely used optical modalities for exogenous contrast‐free, high‐resolution, three‐dimensional imaging in non‐fluorescent scattering tissues. However, sample motion remains a critical barrier to raster‐scanned acquisition and reconstruction of wide‐field anatomically accurate volumetric datasets. We introduce spectrally encoded coherence tomography and reflectometry (SECTR), a high‐speed, multimodality system for simultaneous OCT and spectrally encoded reflectance (SER) imaging. SECTR utilizes a robust system design consisting of shared optical relays, scanning mirrors, swept laser and digitizer to achieve the fastest reported in vivo multimodal imaging rate of 2 gigapixels per second. Our optical design and acquisition scheme enable spatiotemporally co‐registered acquisition of OCT cross‐sections simultaneously with en face SER images for multivolumetric mosaicking. Complementary axial and lateral translation and rotation are extracted from OCT and SER data, respectively, for full volumetric estimation of sample motion with micron spatial and millisecond temporal resolution.      

Overexpression of myocilin in cultured human trabecular meshwork cells

The trabecular meshwork, a specialized eye tissue, is a major site for regulation of the aqueous humor outflow. Malfunctioning of the trabecular meshwork is believed to be responsible for development of glaucoma, a blinding disease. Myocilin is a gene linked to the most common form of glaucoma. Its expression is known to be upregulated by glucocorticoids in trabecular meshwork cells and the altered myocilin level may be the culprit for glaucomatous conditions such as corticosteroid-induced glaucoma. In this study, we examined the influence of myocilin overexpression on the adhesion, spreading, migration, phagocytosis, and apoptosis of human trabecular meshwork cells in culture. When the myocilin expression was increased by 3- to 4-fold, the transfectants showed a dramatic loss of actin stress fibers and focal adhesions. Cell adhesion to fibronectin and spreading were also compromised. Myocilin thus appeared to have a de-adhesive activity, similar to that reported extensively with matricellular proteins. The transfected cells in addition displayed an increased sensitivity to apoptosis. These results demonstrate that overexpression of myocilin renders trabecular meshwork cells in a de-adhesive and vulnerable state. This vulnerability may be the basis for pathologic consequences in subtypes of glaucoma.     

Trabecular meshwork gene expression after selective laser trabeculoplasty

Background

Trabecular meshwork and Schlemm''s canal are the tissues appointed to modulate the aqueous humour outflow from the anterior chamber. The impairment of their functions drives to an intraocular pressure increase. The selective laser trabeculoplasty is a laser therapy of the trabecular meshwork able to decrease intraocular pressure. The exact response mechanism to this treatment has not been clearly delineated yet. The herein presented study is aimed at studying the gene expression changes induced in trabecular meshwork cells by selective laser trabeculoplasty (SLT) in order to better understand the mechanisms subtending its efficacy.

Methodology/Principal Findings

Primary human trabecular meshwork cells cultured in fibroblast medium underwent selective laser trabeculoplasty treatment. RNA was extracted from a pool of cells 30 minutes after treatment while the remaining cells were further cultured and RNA was extracted respectively 2 and 6 hours after treatment. Control cells stored in incubator in absence of SLT treatment were used as reference samples. Gene expression was evaluated by hybridization on miRNA-microarray and laser scanner analysis. Scanning electron microscopic examination was performed on 2 Trabecular meshwork samples after SLT at 4^th and 6^th hour from treatment. On the whole, selective laser trabeculoplasty modulates in trabecular meshwork the expression of genes involved in cell motility, intercellular connections, extracellular matrix production, protein repair, DNA repair, membrane repair, reactive oxygen species production, glutamate toxicity, antioxidant activities, and inflammation.

Conclusions/Significance

SLT did not induce any phenotypic alteration in TM samples. TM is a complex tissue possessing a great variety of function pivotal for the active regulation of aqueous humour outflow from the anterior chamber. SLT is able to modulate these functions at the postgenomic molecular level without inducing damage either at molecular or phenotypic levels.     

Volumetric hand‐held optoacoustic angiography as a tool for real‐time screening of dense breast

Existing mammographic screening solutions are generally associated with several major drawbacks, such as exposure to ionizing radiation or insufficient sensitivity in younger populations with radiographically‐dense breast. Even when combined with ultrasound or magnetic resonance imaging, X‐Ray mammography may still attain unspecific or false positive results. Thus, development of new breast imaging tools represents a timely medical challenge. We report on a new approach to high‐resolution functional and anatomical breast angiography using volumetric hand‐held optoacoustic tomography, which employs light intensities safe for human use. Experiments in young healthy volunteers with fibroglandular‐dominated dense breasts revealed the feasibility of rendering three‐dimensional images representing vascular anatomy and functional blood oxygenation parameters at video rate. Sufficient contrast was achieved at depths beyond 2 cm within dense breasts without compromising the real‐time imaging performance. The suggested solution may thus find applicability as a standalone or supplemental screening tool for early detection and follow‐up of carcinomas in radiographically‐dense breasts.

Volumetric handheld optoacoustic tomography scanner uses safe pulses of near‐infrared light to render three‐dimensional images of deep vascular anatomy, blood oxygenation and breast parenchyma at video rate.     

Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue

Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.     

Mitochondrial damage in the trabecular meshwork occurs only in primary open-angle glaucoma and in pseudoexfoliative glaucoma

Background

Open-angle glaucoma appears to be induced by the malfunction of the trabecular meshwork cells due to injury induced by oxidative damage and mitochondrial impairment. Here, we report that, in fact, we have detected mitochondrial damage only in primary open-angle glaucoma and pseudo-exfoliation glaucoma, among several glaucoma types compared.

Methodology/Principal Findings

Mitochondrial damage was evaluated by analyzing the common mitochondrial DNA deletion by real-time PCR in trabecular meshwork specimens collected at surgery from glaucomatous patients and controls. Glaucomatous patients included 38 patients affected by various glaucoma types: primary open-angle, pigmented, juvenile, congenital, pseudoexfoliative, acute, neovascular, and chronic closed-angle glaucoma. As control samples, we used 16 specimens collected from glaucoma-free corneal donors. Only primary open-angle glaucoma (3.0-fold) and pseudoexfoliative glaucoma (6.3-fold) showed significant increases in the amount of mitochondrial DNA deletion. In all other cases, deletion was similar to controls.

Conclusions/Significance

Despite the fact that the trabecular meshwork is the most important tissue in the physiopathology of aqueous humor outflow in all glaucoma types, the present study provides new information regarding basic physiopathology of this tissue: only in primary open-angle and pseudoexfoliative glaucomas oxidative damage arising from mitochondrial failure play a role in the functional decay of trabecular meshwork.     

Alteration of cytoskeletal structure, integrin distribution, and migratory activity by phagocytic challenge in cells from an ocular tissue—The trabecular meshwork

Summary The trabecular meshwork is a specialized tissue in the anterior chamber of the eye that regulates the aqueous humor outflow and controls the intraocular pressure. Cells in the trabecular meshwork are believed to be essential for maintenance of the outflow system, and their malfunctioning may lead to elevation of intraocular pressure and development of glaucoma. These cells are avid phagocytes. Using an in vitro tissue culture system, we have previously shown that bovine trabecular meshwork cells exhibited a short-term loss of cell-matrix adhesiveness after exposure to latex microspheres. The current study showed that 4 h after phagocytosis, the cytoskeletal structure in trabecular meshwork cells was disrupted, the formation of focal contact formation was limited, and the cellular migratory activity was increased. These in vitro responses paralleled those that occur in vivo. By 24 h, all the changes demonstrated returned to normal. Our data suggest that the short-term loss in cell-matrix cohesiveness observed after phagocytic challenge may be related to the reorganization of cytoskeletal structures and the decline of focal contact formation. The altered cell migration may also be interlinked.     

In vivo blind‐deconvolution photoacoustic ophthalmoscopy with total variation regularization

Photoacoustic ophthalmoscopy (PAOM) is capable of noninvasively imaging anatomic and functional information of the retina in living rodents. However, the strong ocular aberration in rodent eyes and limited ultrasonic detection sensitivity affect PAOM's spatial resolution and signal‐to‐noise ratio (SNR) in in vivo eyes. In this work, we report a computational approach to combine blind deconvolution (BD) algorithm with a regularizing constraint based on total variation (BDTV) for PAOM imaging restoration. We tested the algorithm in retinal and choroidal microvascular images in albino rat eyes. The algorithm improved PAOM's lateral resolution by around 2‐fold. Moreover, it enabled the improvement in imaging SNR for both major vessels and capillaries, and realized the well‐preserved blood vessels' edges simultaneously, which surpasses conventional Richardson‐Lucy BD algorithm. The reported results indicate that the BDTV algorithm potentially facilitate PAOM in extracting retinal pathophysiological information by enhancing in vivo imaging quality without physically modifying PAOM's optical configuration.      

Characterization and application of porous gold nanoparticles as 2‐photon luminescence imaging agents: 20‐fold brighter than gold nanorods

Two‐photon nonlinear microscopy with the aid of plasmonic contrast agents is an attractive bioimaging technique capable of generating high‐resolution images in 3 dimensions and facilitating targeted imaging with deep tissue penetration. In this work, physically synthesized gold nanoparticles containing multiple nanopores are used as 2‐photon contrast agents and are reported to emit a 20‐fold brighter 2‐photon luminescence as compared to typical contrast agents, that is, gold nanorods. A successful application of our porous gold nanoparticles is experimentally demonstrated by in vitro nonlinear optical imaging of adipocytes at subcellular level.      

Intraorbital cerebrospinal fluid outflow and the posterior uveal compartment of the hamster eye

Summary An ultrastructural and tracer study was undertaken to determine normal outflow pathways of cerebrospinal fluid (CSF) at the terminal subarachnoid space (SAS) of the optic nerve. In the morphological studies, the optic nerve dura and arachnoid were found to be continuous with the sclera of the eye beyond the optic nerve SAS. The pia mater is continuous with the inner sciera and the lamina fusca of the eye. Montages and serial sections demonstrated that the distal SAS is divided into numerous tortuous channels to form an arachnoidal trabecular meshwork. Spaces of this meshwork continue into microcanals which bypass the outer arachnoid barrier layers of the optic nerve meninges to reach the sclera and posterior intraorbital connective tissue. Ferritin infused into the cisterna magna entered the optic nerve SAS within 1 min and reached arachnoidal trabecular meshwork channels and the microcanals within 8 min. It then passed into intraorbital connective tissue spaces at the posterior pole of the eye. Ferritin appeared to be blocked by the lamina fusca and a newly discovered posterior compact zone which together prevented its entrance into the choroidal interstitium. These observations suggest that a subarachnoidal-scleral-orbital outflow pathway provides a route for CSF drainage from the optic nerve SAS to intraorbital connective tissue. The previously described posterior uveal compartment in the hamster eye (Kelly et al. 1983) appears to be relatively isolated from this subarachnoidal-scleral-orbital CSF outflow.Parts of this work have been presented at the 1984 meetings of the American Association of Anatomists (Shen 1984).     

Ascorbic acid modulates collagen type I gene expression by cells from an eye tissue--trabecular meshwork.

The trabecular meshwork, a specialized tissue in the anterior chamber of the eye, plays a major role in the regulation of aqueous humor outflow. We studied the effects of ascorbic acid, a significant component in the aqueous humor, on gene expression of type I collagen in cultures of bovine trabecular meshwork cells. These cells were plated for 6 days, exposed to ascorbic acid in concentrations of 100, 250 and 500 micrograms/ml for 3 days and labeled with (3H)proline for the last 24 hrs. Cultures that did not receive ascorbic acid served as controls. Bacterial collagenase assays showed enhanced incorporation of (3H)proline into collagenous proteins in cultures treated with 100 and 250 micrograms/ml of ascorbic acid. Gel electrophoresis and fluorography revealed that ascorbic acid caused a 2.6- to 4.9-fold increase in production of alpha 1 (I) and alpha 2(I) collagen chains by trabecular meshwork cells. Such an increase was found, using a cDNA probe specific for pro alpha 1(I) chains, to be accompanied by an increase in steady-state mRNA levels. Similar findings were also yielded from in situ hybridization experiments. These results, coupled with previously demonstrated ascorbate-induced effects on glycosaminoglycan, fibronectin and laminin synthesis, suggest that ascorbic acid is a key mediator of the extracellular matrix production by trabecular meshwork cells. Fluctuations in its concentration may lead to alterations in the makeup and assembly of matrices underlying the cells.     

Isotropic high resolution optoacoustic imaging with linear detector arrays in bi‐directional scanning

Optoacoustic (photoacoustic) imaging is often performed with one‐dimensional transducer arrays, in analogy to ultrasound imaging. Optoacoustic imaging using linear arrays offers ease of implementation but comes with several performance drawbacks, in particular poor elevation resolution, i.e. the resolution along the axis perpendicular to the focal plane. Herein, we introduce and investigate a bi‐directional scanning approach using linear arrays that can improve the imaging performance to quasi‐isotropic transverse resolution. We study the approach theoretically and perform numerical simulations and phantom measurements to evaluate its performance under defined conditions. Finally, we discuss the features and the limitations of the proposed method.

The poor elevation resolution in a linear scan (left image) is overcome by the proposed bi‐directional scanning approach that yields isotropic transverse resolution (right).     

Mechanisms of ATP release, the enabling step in purinergic dynamics

The only effective intervention to slow onset and progression of glaucomatous blindness is to lower intraocular pressure (IOP). Among other modulators, adenosine receptors (ARs) exert complex regulation of IOP. Agonists of A(3)ARs in the ciliary epithelium activate Cl(-) channels, favoring increased formation of aqueous humor and elevated IOP. In contrast, stimulating A(1)ARs in the trabecular outflow pathway enhances release of matrix metalloproteinases (MMPs) from trabecular meshwork (TM) cells, reducing resistance to outflow of aqueous humor to lower IOP. These opposing actions are thought to be initiated by cellular release of ATP and its ectoenzymatic conversion to adenosine. This view is now supported by our identification of six ectoATPases in trabecular meshwork (TM) cells and by our observation that external ATP enhances TM-cell secretion of MMPs through ectoenzymatic formation of adenosine. ATP release is enhanced by cell swelling and stretch. Also, enhanced ATP release and downstream MMP secretion is one mediator of the action of actin depolymerization to reduce outflow resistance. Inflow and outflow cells share pannexin-1 and connexin hemichannel pathways for ATP release. However, vesicular release and P2X(7) release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering IOP.     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

Aqueous Angiography: Real-Time and Physiologic Aqueous Humor Outflow Imaging

Purpose

Trabecular meshwork (TM) bypass surgeries attempt to enhance aqueous humor outflow (AHO) to lower intraocular pressure (IOP). While TM bypass results are promising, inconsistent success is seen. One hypothesis for this variability rests upon segmental (non-360 degrees uniform) AHO. We describe aqueous angiography as a real-time and physiologic AHO imaging technique in model eyes as a way to simulate live AHO imaging.

Methods

Pig (n = 46) and human (n = 6) enucleated eyes were obtained, orientated based upon inferior oblique insertion, and pre-perfused with balanced salt solution via a Lewicky AC maintainer through a 1mm side-port. Fluorescein (2.5%) was introduced intracamerally at 10 or 30 mm Hg. With an angiographer, infrared and fluorescent (486 nm) images were acquired. Image processing allowed for collection of pixel information based on intensity or location for statistical analyses. Concurrent OCT was performed, and fixable fluorescent dextrans were introduced into the eye for histological analysis of angiographically active areas.

Results

Aqueous angiography yielded high quality images with segmental patterns (p<0.0001; Kruskal-Wallis test). No single quadrant was consistently identified as the primary quadrant of angiographic signal (p = 0.06–0.86; Kruskal-Wallis test). Regions of high proximal signal did not necessarily correlate with regions of high distal signal. Angiographically positive but not negative areas demonstrated intrascleral lumens on OCT images. Aqueous angiography with fluorescent dextrans led to their trapping in AHO pathways.

Conclusions

Aqueous angiography is a real-time and physiologic AHO imaging technique in model eyes.     

Miniature probe for dual‐modality photoacoustic microscopy and white‐light microscopy for image guidance: A prototype toward an endoscope

In this study, a novel photoacoustic microscopy (PAM) probe integrating white‐light microscopy (WLM) modality that provides guidance for PAM imaging and complementary information is implemented. One single core of an imaging fiber bundle is employed to deliver a pulsed laser for photoacoustic excitation for PAM mode, which provides high resolution with deep penetration. Meanwhile, for WLM mode, the imaging fiber bundle is used to transmit two‐dimensional superficial images. Lateral resolution of 7.2 μm for PAM is achieved. Since miniature components are used, the probe diameter is only 1.7 mm. Imaging of phantom and animals in vivo is conducted to show the imaging capability of the probe. The probe has several advantages by introducing the WLM mode, such as being able to conveniently identify regions of interest and align the focus for PAM mode. The prototype of an endoscope shows potential to facilitate clinical photoacoustic endoscopic applications.     

Front Cover: Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second (J. Biophotonics 4/2018)

SECTR is a novel multimodal imaging platform for combined volumetric optical coherence tomography (OCT) and en face spectrally encoded reflectometry (SER). The authors demonstrate three‐dimensional motion‐tracking with millisecond temporal and micron spatial resolution using complementary data from OCT and SER, and preliminary algorithms and results showing real‐time image aiming and multi‐volumetric mosaicking for reconstruction of wide‐field composites. The image shows a noninvasively imaged nine‐field mosaic of in vivo human retina and depth‐resolved visualization of tissue microstructures. Further details can be found in the article by Mohamed T. El‐Haddad, Ivan Bozic, and Yuankai K. Tao ( e201700268 )

     

Multiparametric evaluation of hindlimb ischemia using time‐series indocyanine green fluorescence imaging

Peripheral arterial disease (PAD) can further cause lower limb ischemia. Quantitative evaluation of the vascular perfusion in the ischemic limb contributes to diagnosis of PAD and preclinical development of new drug. In vivo time‐series indocyanine green (ICG) fluorescence imaging can noninvasively monitor blood flow and has a deep tissue penetration. The perfusion rate estimated from the time‐series ICG images is not enough for the evaluation of hindlimb ischemia. The information relevant to the vascular density is also important, because angiogenesis is an essential mechanism for post‐ischemic recovery. In this paper, a multiparametric evaluation method is proposed for simultaneous estimation of multiple vascular perfusion parameters, including not only the perfusion rate but also the vascular perfusion density and the time‐varying ICG concentration in veins. The target method is based on a mathematical model of ICG pharmacokinetics in the mouse hindlimb. The regression analysis performed on the time‐series ICG images obtained from a dynamic reflectance fluorescence imaging system. The results demonstrate that the estimated multiple parameters are effective to quantitatively evaluate the vascular perfusion and distinguish hypo‐perfused tissues from well‐perfused tissues in the mouse hindlimb. The proposed multiparametric evaluation method could be useful for PAD diagnosis.

The estimated perfusion rate and vascular perfusion density maps (left) and the time‐varying ICG concentration in veins of the ankle region (right) of the normal and ischemic hindlimbs.     

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C₆ symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.