A2E Distribution in RPE Granules in Human Eyes.

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1-F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.

Optical coherence photoacoustic microscopy for in vivo multimodal retinal imaging.

We developed an optical coherence photoacoustic microscopy (OC-PAM) system, which can accomplish optical coherence tomography (OCT) and photoacoustic microscopy (PAM) simultaneously by using a single pulsed broadband light source. With a center wavelength of 800 nm and a bandwidth of 30 nm, the system is suitable for imaging the retina. Generated from the same group of photons, the OCT and PAM images are intrinsically registered in the lateral directions. To test the capabilities of the system on multimodal ophthalmic imaging, we imaged the retina of pigmented rats. The OCT images showed the retinal structures with quality similar to conventional OCT, while the PAM images revealed the distribution of absorbers in the retina. Since the absorption of hemoglobin is relatively weak at around 800 nm, the NIR PAM signals are generated mainly from melanin in the posterior segment of the eye, thus providing melanin-specific imaging of the retina.

Regenerative potential of the zebrafish corneal endothelium.

Corneal transparency, critical for clear vision, is maintained in part by the pump function of the corneal endothelial cells that are arrested in G(1) phase of the cell cycle in adult humans. Thus loss of endothelial cells leads to a decrease in endothelial cell density. A decrease below a critical threshold results in corneal edema and subsequent vision loss. Corneal edema due to endothelial dysfunction is a common indication for transplantation in developed countries. The zebrafish has emerged as a model for vertebrate regeneration due to its ease of genetic manipulation and remarkable regenerative capacity. The purpose of this study was to investigate the response and regenerative potential of the zebrafish corneal endothelium to pharmacological and mechanical injury. Similar to the human cornea, Na(+)/K(+) ATPase activity is necessary to maintain the pump function as intracameral injection of ouabain resulted in an increase in central corneal thickness. Surgical removal of the majority of the central corneal endothelium resulted in a similar increase in corneal thickness. Remarkably, by just one week post-injury the central corneal endothelium had largely re-formed. Immunofluorescence of phosphorylated histone H3 indicated that this recovery correlated with corneal endothelial cells re-entering the cell cycle. In conclusion, our results establish zebrafish as a useful model of corneal injury and repair that may offer insights into the mechanism of cell cycle arrest in human corneal endothelial cells.

Optical coherence tomography for whole eye segment imaging.

We proposed a dual focus dual channel spectral domain optical coherence tomography (SD-OCT) for simultaneous imaging of the whole eye segments from cornea to the retina. By using dual channels the system solved the problem of limited imaging depth of SD-OCT. By using dual focus the system solved the problem of simultaneous light focusing on the anterior segment of the eye and the retina. Dual focusing was achieved by adjusting the collimating lenses so the divergence of the two probing beams was tuned to make them focused at different depth in the eye. We further achieved full range complex (FRC) SD-OCT in one channel to increase the depth range for anterior segment imaging. The system was successfully tested by imaging a human eye in vivo.

Regeneration pattern and genome-wide transcription profile of rhizome axillary buds after perennial rice harvest.

Perennial rice is a new type of rice that allows the harvest of rice for multiple years without growing new seedlings annually. This technology represents a green and sustainable agricultural production mode with many advantages for balancing agricultural ecology and food security. However, the differences in regeneration patterns between perennial and annual rice and the gene regulatory pathways of the apical dominance in axillary bud growth after harvest in perennial rice are still unclear. In this study, perennial rice (PR23) and annual rice (Chugeng28) were used to investigate axillary bud growth patterns before and after apical spike removal. After elimination of apical dominance at different development stages, perennial rice rhizome axillary buds at the compression nodes germinated more rapidly than others and developed into new seedlings. The axillary buds at the high-position nodes in annual rice grew faster than those at other nodes. Furthermore, the global gene expression patterns of PR23 rhizome buds at compression nodes grown for 1, 3, 4, and 5 days after apical spike removal were analyzed by transcriptome sequencing. Compared with the control buds without apical removal, 264, 3,484, 2,095, and 3,398 genes were up-regulated, and 674, 3,484, 1,594, and 1,824 genes were down-regulated in the buds grown for 1, 3, 4, and 5 days after apical spike removal, respectively. Trend analysis of the expressed genes at different time points was performed and co-expression network was constructed to identify key genes in rhizome axillary bud regrowth. The results showed that 85 hub genes involved in 12 co-regulatory networks were mainly enriched in the light system, photosynthesis-antenna protein, plant hormone signal transduction, ABC transporter and metabolic pathways, which suggested that hormone and photosynthetic signals might play important roles in the regulation of rhizome axillary bud regeneration in perennial rice. Overall, this study clarified the differences in the regeneration patterns of axillary buds between perennial and annual rice and provided insight into the complex regulatory networks during the regeneration of rhizome axillary buds in perennial rice.

σ2R/TMEM97 in retinal ganglion cell degeneration.

The sigma 2 receptor (σ2R) was recently identified as an endoplasmic reticulum (ER) membrane protein known as transmembrane protein 97 (TMEM97). Studies have shown that σ2R/TMEM97 binding compounds are neuroprotective, suggesting a role of σ2R/TMEM97 in neurodegenerative processes. To understand the function of σ2R/TMEM97 in neurodegeneration pathways, we characterized ischemia-induced retinal ganglion cell (RGC) degeneration in TMEM97-/- mice and found that RGCs in TMEM97-/- mice are resistant to degeneration. In addition, intravitreal injection of a selective σ2R/TMEM97 ligand DKR-1677 significantly protects RGCs from ischemia-induced degeneration in wildtype mice. Our results provide conclusive evidence that σ2R/TMEM97 plays a role to facilitate RGC death following ischemic injury and that inhibiting the function of σ2R/TMEM97 is neuroprotective. This work is a breakthrough toward elucidating the biology and function of σ2R/TMEM97 in RGCs and likely in other σ2R/TMEM97 expressing neurons. Moreover, these findings support future studies to develop new neuroprotective approaches for RGC degenerative diseases by inhibiting σ2R/TMEM97.

Dual-band spectral-domain optical coherence tomography for in vivo imaging the spectral contrasts of the retinal nerve fiber layer.

The ultimate goal of the study is to provide an imaging tool to detect the earliest signs of glaucoma before clinically visible damage occurs to the retinal nerve fiber layer (RNFL). Studies have shown that the optical reflectance of the damaged RNFL at short wavelength (<560 nm) is reduced much more than that at long wavelength, which provides spectral contrast for imaging the earliest damage to the RNFL. To image the spectral contrast we built a dual-band spectral-domain optical coherence tomography (SD-OCT) centered at 808 nm (NIR) and 415 nm (VIS). The light at the two bands was provided by the fundamental and frequency-doubled outputs of a broadband Ti:Sapphire laser. The depth resolution of the NIR and VIS OCT systems are 4.7 µm and 12.2 µm in the air, respectively. The system was applied to imaging the rat retina in vivo. Significantly different appearances between the OCT cross sectional images at the two bands were observed. The ratio of the light reflected from the RNFL over that reflected from the entire retina at the two bands were quantitatively compared. The experimental results showed that the dual-band OCT system is feasible for imaging the spectral contrasts of the RNFL.

Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging.

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.

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.

Use of ultra-high-resolution optical coherence tomography to detect in vivo characteristics of Descemet's membrane in Fuchs' dystrophy.

PURPOSE: To demonstrate the capability of ultra-high-resolution (UHR) anterior segment optical coherence tomography (OCT) to image Descemet's membrane (DM) and measure its thickness in vivo. (2) To evaluate the use of DM characteristics and thickness in the diagnosis of Fuchs' dystrophy. DESIGN: Case-control study. PARTICIPANTS: Twenty eyes of 12 Fuchs' dystrophy patients, 20 eyes of 13 young normal, and 20 eyes of 15 elderly normal subjects. METHODS: Subjects were imaged using novel, custom-built UHR-OCT. Images were used to describe the characteristics of DM. Custom-made software was used to measure DM thickness and central corneal thickness (CCT). Specimens of DM obtained from Fuchs' dystrophy patients who underwent endothelial keratoplasty (EK) were histopathologically examined. Regression analyses were used to assess the correlation of DM thickness measured by UHR-OCT in vivo and by light microscopy and to determine the intergroup correlations between age, CCT, and DM thickness. MAIN OUTCOME MEASURES: We assessed DM characteristics and thickness, CCT, and age. RESULTS: Using UHR-OCT, the DM seemed in normal young subjects as a single, opaque, smooth line and in normal elderly subjects as a band of 2 smooth opaque lines with a translucent space in between. In Fuchs' dystrophy, DM appeared as a thickened band of 2 opaque lines; the anterior line was smooth whereas the posterior line had a wavy and irregular appearance with areas of localized thickenings. The DM thickness measured in vivo by UHR-OCT correlated significantly with that measured by light microscopy in 5 Fuchs' dystrophy eyes that underwent EK. The average central thicknesses of DM in normal young, in normal elderly and in Fuchs' dystrophy eyes were 10+/-3, 16+/-2, and 34+/-11 microm, respectively (P<0.001). There was a significant correlation between age and DM thickness only in normal groups. In Fuchs' dystrophy patients, there was a significant correlation between CCT and DM thickness that was not significant for normal groups. CONCLUSIONS: Ultra-high-resolution OCT is an innovative technique for the in vivo imaging of DM. Determining DM characteristics and thickness by UHR-OCT could be a new approach for the diagnosis of Fuchs' dystrophy.

Collecting back-reflected photons in photoacoustic microscopy.

Since the photoacoustic effect relies only on the absorbed optical energy, the back-reflected photons from samples in optical-resolution photoacoustic microscopy are usually discarded. By employing a 2 x 2 single-mode fiber optical coupler in a laser-scanning optical-resolution photoacoustic microscope for delivering the illuminating laser light and collecting the back reflected photons, a fiber-optic confocal microscope is integrated with the photoacoustic microscope. Thus, simultaneous multimodal imaging can be achieved with a single light source and images from the two modalities are intrinsically registered. Such capabilities are demonstrated in imaging both phantoms and small animals in vivo.

Adaptive optics photoacoustic microscopy.

We have developed an adaptive optics photoacoustic microscope (AO-PAM) for high-resolution imaging of biological tissues, especially the retina. To demonstrate the feasibility of AO-PAM we first designed the AO system to correct the wavefront errors of the illuminating light of PAM. The aberrations of the optical system delivering the illuminating light to the sample in PAM was corrected with a close-loop AO system consisting of a 141-element MEMS-based deformable mirror (DM) and a Shack-Hartmann (SH) wavefront sensor operating at 15 Hz. The photoacoustic signal induced by the illuminating laser beam was detected by a custom-built needle ultrasonic transducer. When the wavefront errors were corrected by the AO system, the lateral resolution of PAM was measured to be better than 2.5 µm using a low NA objective lens. We tested the system on imaging ex vivo ocular samples, e.g., the ciliary body and retinal pigment epithelium (RPE) of a pig eye. The AO-PAM images showed significant quality improvement. For the first time we were able to resolve single RPE cells with PAM.

Photoacoustic ophthalmoscopy for in vivo retinal imaging.

We have developed a non-invasive photoacoustic ophthalmoscopy (PAOM) for in vivo retinal imaging. PAOM detects the photoacoustic signal induced by pulsed laser light shined onto the retina. By using a stationary ultrasonic transducer in contact with the eyelids and scanning only the laser light across the retina, PAOM provides volumetric imaging of the retinal micro-vasculature and retinal pigment epithelium at a high speed. For B-scan frames containing 256 A-lines, the current PAOM has a frame rate of 93 Hz, which is comparable with state-of-the-art commercial spectral-domain optical coherence tomography (SD-OCT). By integrating PAOM with SD-OCT, we further achieved OCT-guided PAOM, which can provide multi-modal retinal imaging simultaneously. The capabilities of this novel technology were demonstrated by imaging both the microanatomy and microvasculature of the rat retina in vivo.

Simultaneous dual molecular contrasts provided by the absorbed photons in photoacoustic microscopy.

We investigated the feasibility of simultaneously imaging two distinctive molecular contrasts provided by the absorbed photons in biological tissues with a single light source. The molecular contrasts are based on two physical effects induced by the absorbed photons: photoacoustics (PA) and autofluorescence (AF). In an integrated multimodal imaging system, the PA and AF signals were detected by a high-sensitivity ultrasonic transducer and an avalanche photodetector, respectively. The system was tested by imaging ocular tissue samples, including the retinal pigment epithelium and the ciliary body. The acquired images provided information on the spatial distributions of melanin and lipofuscin in these samples.

Quantifying lipofuscin in retinal pigment epithelium in vivo by visible-light optical coherence tomography-based multimodal imaging.

Lipofuscin in the retinal pigment epithelium (RPE) is the major source of fundus autofluorescence (FAF). A technical challenge to accurately quantify the FAF intensities, thus the lipofuscin concentration, is to compensate the light attenuation of RPE melanin. We developed the VIS-OCT-FAF technology to accomplish optical coherence tomography (OCT) and FAF simultaneously with a single broadband visible light source. We demonstrated that light attenuation by RPE melanin can be assessed and corrected using the depth-resolved OCT signals. FAF images from albino and pigmented rats showed that without compensation, FAF signals from pigmented rats are lower than that from albinos. After compensation, however, FAF signals from pigmented rats are higher. This finding is supported by measurements of lipofuscin fluorophore A2E in the RPE using liquid chromatography/mass spectrometry (LC/MS) showing that compensated FAF intensities correlate linearly with A2E contents. The present work represents an important step toward accurately assessing RPE lipofuscin concentrations by FAF.

Dual channel dual focus optical coherence tomography for imaging accommodation of the eye.

A dual channel dual focus spectral-domain optical coherence tomography was developed for imaging the accommodation of the eye in real time. The system can provide simultaneous cross-sectional imaging of all the surfaces of the anterior segment of the eye including the cornea, anterior chamber, anterior and posterior surfaces of the crystalline lens. Thus, the modification of the curvatures of the anterior and posterior surfaces of the crystalline lens and the dimensions of the anterior segment of the eye with accommodation can be calculated. The system was successfully tested in imaging accommodation. The preliminary results demonstrated the feasibility of this novel approach.

Retinal tumor imaging and volume quantification in mouse model using spectral-domain optical coherence tomography.

We have successfully imaged the retinal tumor in a mouse model using an ultra-high resolution spectral-domain optical coherence tomography (SD-OCT) designed for small animal retinal imaging. For segmentation of the tumor boundaries and calculation of the tumor volume, we developed a novel segmentation algorithm. The algorithm is based on parametric deformable models (active contours) and is driven by machine learning-based region classification, namely a Conditional Random Field. With this algorithm we are able to obtain the tumor boundaries automatically, while the user can specify additional constraints (points on the boundary) to correct the segmentation result, if needed. The system and algorithm were successfully applied to studies on retinal tumor progression and monitoring treatment effects quantitatively in a mouse model of retinoblastoma.

Simultaneous multimodal imaging with integrated photoacoustic microscopy and optical coherence tomography.

We have developed a multimodal imaging technique by integrating photoacoustic microscopy and spectral-domain optical coherence tomography to provide simultaneous volumetric microscopic imaging of both optical absorption and scattering contrasts in biological tissues. In the integrated system, the two imaging modalities share the same optical scanning and delivery mechanisms after their probing and illumination light beams are combined. By further synchronizing the image acquisitions, the images from the two modalities are intrinsically registered. The capabilities of this novel technique were demonstrated by imaging both the microanatomy and microvasculature in mouse ears in vivo.

Ultra-high resolution spectral domain optical coherence tomography of traumatic maculopathy.

The authors describe a patient who suffered traumatic maculopathy following blunt trauma to the eye with commotio retinae, subretinal and preretinal hemorrhage, traumatic macular hole, and outer retinal and retinal pigment epithelium disruption. Serial imaging with commercially available and ultra-high resolution spectral domain optical coherence tomography characterized the evolution of the injury.

In situ visualization of tears on contact lens using ultra high resolution optical coherence tomography.

OBJECTIVE: To demonstrate the capability of directly visualizing the tear film on contact lenses using optical coherence tomography (OCT). METHODS: Six eyes of three healthy subjects wearing PureVision and ACUVUE Advance soft and Boston RGP hard contact lenses were imaged with a custom built, high speed, ultra-high resolution spectral domain optical coherence tomograph. Refresh Liquigel was used to demonstrate the effect of artificial tears on the tear film. RESULTS: Ultra high resolution images of the pre- and post-lens films were directly visualized when each lens was inserted onto the eye. After the instillation of artificial tears during lens wear, the tear film was thicker. The post-lens tear film underneath the lens edge was clearly shown. Interactions between the lens edges and the ocular surface were obtained for each of the lens types and base curves. With a contrast enhancement agent, tear menisci on the contact lenses around the upper and lower eyelids were highlighted. With hard contact lenses, the tear film was visualized clearly and changed after a blink when the lens was pulled up by the lid. CONCLUSIONS: Ultra-high resolution OCT is a potentially promising technique for imaging tears around contact lenses. This successful demonstration of in situ post-lens tear film imaging suggests that OCT could open a new era in studying tear dynamics during contact lens wear. The novel method may lead to new ways of evaluating contact lens fitting.