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.

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.

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.

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.

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.

Optical coherence tomography-guided dynamic focusing for combined optical and mechanical scanning multimodal photoacoustic microscopy.

To achieve fast imaging and large field of view (FOV), we improved our multimodal imaging system, which integrated optical resolution photoacoustic microscopy, optical coherence tomography (OCT), and confocal fluorescence microscopy in one platform, by combining optical scanning with mechanical scanning. To ensure good focusing of the objective lens over all the imaged area, we employed OCT-guided dynamic focusing. Different from our previous point-by-point dynamic focusing, we employed an area-by-area focusing adjustment strategy, in which each fast optical scanning area has a fixed focusing depth. We have demonstrated the performance of the system by imaging biological samples ex vivo (plant leaf) and in vivo (mouse ear). The system has achieved uniform resolution in an FOV of 10 mm × 10 mm with an imaging time of about 5 min.

Integrated multimodal photoacoustic microscopy with OCT- guided dynamic focusing.

Combining different contrast mechanisms to achieve simultaneous multimodal imaging is always desirable but is challenging due to the various optical and hardware requirements for different imaging systems. We developed a multimodal microscopic optical imaging system with the capability of providing comprehensive structural, functional and molecular information of living tissues. This imaging system integrated photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT) and confocal fluorescence microscopy in one platform. By taking advantage of the depth resolving capability of OCT, we developed a novel OCT-guided surface contour scanning methodology for dynamic focusing adjustment. We have conducted phantom, in vivo, and ex vivo tests to demonstrate the capability of the multimodal imaging system for providing comprehensive microscopic information of biological tissues. Integrating all the aforementioned imaging modalities with OCT-guided dynamic focusing for simultaneous multimodal imaging has promising potential for preclinical research and clinical practice in the future.

Visible-light optical coherence tomography-based multimodal system for quantitative fundus autofluorescence imaging.

IMPACT STATEMENT: Quantitative fundus autofluorescence imaging with simultaneous visible-light optical coherence tomography-based multimodal technology has potential significant impact on the diagnosis and monitoring the progression of retinal diseases.

Visible light OCT-based quantitative imaging of lipofuscin in the retinal pigment epithelium with standard reference targets.

We developed a technology for quantitative retinal autofluorescence (AF, or FAF for fundus AF) imaging for quantifying lipofuscin in the retinal pigment epithelium (RPE). The technology is based on simultaneous visible light optical coherence tomography (VIS-OCT) and AF imaging of the retina and a pair of reference standard targets at the intermediate retinal imaging plane with known reflectivity for the OCT and fluorescence efficiency for the FAF. The technology is able to eliminate the pre-RPE attenuation in FAF imaging by using the simultaneously acquired VIS-OCT image. With the OCT and fluorescence images of the reference targets, the effects of illumination power and detector sensitivity can be eliminated.

Visible-light optical coherence tomography-based multimodal retinal imaging for improvement of fluorescent intensity quantification.

We developed a spectral-domain visible-light optical coherence tomography (VIS-OCT) based multimodal imaging technique which can accomplish simultaneous OCT and fluorescence imaging with a single broadband light source. Phantom experiments showed that by using the simultaneously acquired OCT images as a reference, the effect of light attenuation on the intensity of the fluorescent images by materials in front of the fluorescent target can be compensated. This capability of the multimodal imaging technique is of high importance for achieving quantification of the true intensities of autofluorescence (AF) imaging of the retina. We applied the technique in retinal imaging including AF imaging of the retinal pigment epithelium and fluorescein angiography (FA). We successfully demonstrated the effect of compensation on AF and FA images with the simultaneously acquired VIS-OCT images.

Depth-resolved rhodopsin molecular contrast imaging for functional assessment of photoreceptors.

Rhodopsin, the light-sensing molecule in the outer segments of rod photoreceptors, is responsible for converting light into neuronal signals in a process known as phototransduction. Rhodopsin is thus a functional biomarker for rod photoreceptors. Here we report a novel technology based on visible-light optical coherence tomography (VIS-OCT) for in vivo molecular imaging of rhodopsin. The depth resolution of OCT allows the visualization of the location where the change of optical absorption occurs and provides a potentially accurate assessment of rhodopsin content by segmentation of the image at the location. Rhodopsin OCT can be used to quantitatively image rhodopsin distribution and thus assess the distribution of functional rod photoreceptors in the retina. Rhodopsin OCT can bring significant impact into ophthalmic clinics by providing a tool for the diagnosis and severity assessment of a variety of retinal conditions.

Measuring retinal blood flow in rats using Doppler optical coherence tomography without knowing eyeball axial length.

PURPOSE: Doppler optical coherence tomography (OCT) is widely used for measuring retinal blood flow. Existing Doppler OCT methods require the eyeball axial length, in which empirical values are usually used. However, variations in the axial length can create a bias unaccounted for in the retinal blood flow measurement. The authors plan to develop a Doppler OCT method that can measure the total retinal blood flow rate without requiring the eyeball axial length. METHODS: The authors measured the retinal blood flow rate using a dual-ring scanning protocol. The small and large scanning rings entered the eye at different incident angles (small ring: 4°; large ring: 6°), focused on different locations on the retina, and detected the projected velocities/phase shifts along the probing beams. The authors calculated the ratio of the projected velocities between the two rings, and then used this ratio to estimate absolute flow velocity. The authors tested this method in both Intralipid phantoms and in vivo rats. RESULTS: In the Intralipid flow phantom experiments, the preset and measured flow rates were consistent with the coefficient of determination as 0.97. Linear fitting between preset and measured flow rates determined the fitting slope as 1.07 and the intercept as -0.28. In in vivo rat experiments, the measured average total retinal blood flow was 7.02 ± 0.31 µl/min among four wild-type rats. The authors' measured flow rates were consistent with results in the literature. CONCLUSIONS: By using a dual-ring scanning protocol with carefully controlled incident angle difference between the two scanning rings in Doppler OCT, the authors demonstrated that it is feasible to measure the absolute retinal blood flow without knowing the eyeball axial length.

Dual band dual focus optical coherence tomography for imaging the whole eye segment.

We developed an improved dual band dual focus spectral domain optical coherence tomography (SD-OCT) for in vivo 2D/3D imaging of the whole eye segment, including the whole anterior segment and retina. The system featured two OCT channels with two different bands centered at 840 nm and 1050 nm, which were designed to image the retina and the anterior segments of the eye, respectively. By combing the two probe light beams for co-axial scanning and separating them for focusing at different segments of the eye with a combination of three dichroic mirrors, we not only minimized the loss of the backscattered light from the sample but also improved the imaging depth, scan range and resolution. The full resolved complex (FRC) method was applied to double the imaging depth for the whole anterior segment imaging, with which an imaging depth of 36.71 mm in air was achieved. We demonstrated that this system was capable of measuring the dynamic changes of ocular dimensions, including the asphericity of the cornea and lens, during accommodation.

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.

In vivo imaging rhodopsin distribution in the photoreceptors with nano-second pulsed scanning laser ophthalmoscopy.

BACKGROUND: Rhodopsin is a biomarker for the function of rod photoreceptors, the dysfunction of which is related to many blinding diseases like retinitis pigmentosa and age-related macular degeneration. Imaging rhodopsin quantitatively may provide a powerful clinical tool for diagnosis of these diseases. To map rhodopsin distribution accurately in the retina, absorption by rhodopsin intermediates need to be minimized. METHODS AND MATERIALS: We developed nano-second pulsed scanning laser ophthalmoscopy (SLO) to image rhodopsin distribution in the retina. The system takes advantage of the light-induced shift of rhodopsin absorption spectra, which in turn affects the fundus spectral reflection before and after photo-bleaching. By imaging the retina twice, one in the dark-adapted state and the other one in the light-adapted state, the rhodopsin absorption change can be calculated from the differential image, which is a function of the rhodopsin concentration in the rod photoreceptors. RESULTS: The system was successfully applied to in vivo imaging of rat retina in different bleaching conditions to verify its feasibility. Our studies showed that the differential image between the dark- and light-adapted states represents rhodopsin distribution in the retina. We also conducted a dynamic bleaching experiment to prove the importance of reducing light absorption of rhodopsin intermediates. CONCLUSIONS: The preliminary results showed that our nano-second pulsed-light SLO is promising in imaging the functional biomarker of the rod photoreceptors. By using nanosecond pulsed laser, in which one laser pulse generates one pixel of the image, the absorption of rhodopsin intermediates can be reduced.

A combined method to quantify the retinal metabolic rate of oxygen using photoacoustic ophthalmoscopy and optical coherence tomography.

Quantitatively determining physiological parameters at a microscopic level in the retina furthers the understanding of the molecular pathways of blinding diseases, such as diabetic retinopathy and glaucoma. An essential parameter, which has yet to be quantified noninvasively, is the retinal oxygen metabolic rate (rMRO2). Quantifying rMRO2 is challenging because two parameters, the blood flow rate and hemoglobin oxygen saturation (sO2), must be measured together. We combined photoacoustic ophthalmoscopy (PAOM) with spectral domain-optical coherence tomography (SD-OCT) to tackle this challenge, in which PAOM measured the sO2 and SD-OCT mapped the blood flow rate. We tested the integrated system on normal wild-type rats, in which the measured rMRO2 was 297.86 ± 70.23 nl/minute. This quantitative method may shed new light on both fundamental research and clinical care in ophthalmology in the future.