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Type 2 diabetes is characterized by β and α cell dysfunction. We used phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) to monitor oxidative phosphorylation and glycolysis in living islet cells before and after glucose stimulation. In healthy cells, glucose enhanced oxidative phosphorylation in β cells and suppressed oxidative phosphorylation in α cells. In Type 2 diabetes, glucose increased glycolysis in β cells, and only partially suppressed oxidative phosphorylation in α cells. FLIM uncovers key perturbations in glucose induced metabolism in living islet cells and provides a sensitive tool for drug discovery in diabetes.

PurposeHuman pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation.MethodsLive hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue.ResultsWe used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae.ConclusionsLive imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form.

The human cytomegalovirus glycoprotein gp68 functions as an Fc receptor for host IgGs and can form antibody bipolar bridging (ABB) complexes in which gp68 binds the Fc region of an antigen-bound IgG. Here we show that gp68-mediated endocytosis transports ABB complexes into endosomes, after which the complex is routed to lysosomes, presumably for degradation. These results suggest gp68 contributes to evasion of IgG-mediated immune responses by mediating destruction of host IgG and viral antigens.

The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.

PurposeTo demonstrate the use of phase-variance optical coherence tomography (PV-OCT) angiography for detection of pigment epithelial detachment (PED) vascularization in age-related macular degeneration (AMD).Patients and methodsPatients with PEDs and exudative AMD were evaluated by the Retina Services at the University of California, Davis, and the University of California, San Francisco. Each subject underwent fluorescein angiography and structural optical coherence tomography (OCT). Phase-variance OCT analysis was used to create angiographic images of the retinal and choroidal vasculature. PV-OCT-generated B-scans were superimposed on structural OCT B-scans to allow easy identification of perfused vascular structures.ResultsThree patients with vascularized PEDs were imaged with PV-OCT, and each was found to have a vascular signal extending from the choroid into the hyperreflective substance of the PED. Two patients who had no evidence of PED vascularization on fluorescein angiography did not have vascular signals within their PEDs on PV-OCT.ConclusionStructural OCT and PV-OCT images can be combined to create composite B-scans that offer high-resolution views of the retinal tissue along with dynamic vascular visualization. This technique offers a fast, noninvasive method for detecting vascularization of PEDs in AMD and may aid in the early detection of neovascular disease.

PurposePhase-variance optical coherence tomography (PV-OCT) provides volumetric imaging of the retinal vasculature without the need for intravenous injection of a fluorophore. We compare images from PV-OCT and fluorescein angiography (FA) for normal individuals and patients with age-related macular degeneration (AMD) and diabetic retinopathy.DesignThis is an evaluation of a diagnostic technology.ParticipantsFour patients underwent comparative retinovascular imaging using FA and PV-OCT. Imaging was performed on 1 normal individual, 1 patient with dry AMD, 1 patient with exudative AMD, and 1 patient with nonproliferative diabetic retinopathy.MethodsFluorescein angiography imaging was performed using a Topcon Corp (Tokyo, Japan) (TRC-50IX) camera with a resolution of 1280 (H) × 1024 (V) pixels. The PV-OCT images were generated by software data processing of the entire cross-sectional image from consecutively acquired B-scans. Bulk axial motion was calculated and corrected for each transverse location, reducing the phase noise introduced from eye motion. Phase variance was calculated through the variance of the motion-corrected phase changes acquired within multiple B-scans at the same position. Repeating these calculations over the entire volumetric scan produced a 3-dimensional PV-OCT representation of the vasculature.Main outcome measuresFeasibility of rendering retinal and choroidal microvasculature using PV-OCT was compared qualitatively with FA, the current gold standard for retinovascular imaging.ResultsPhase-variance OCT noninvasively rendered a 2-dimensional depth color-coded vasculature map of the retinal and choroidal vasculature. The choriocapillaris was imaged with better resolution of microvascular detail using PV-OCT. Areas of geographic atrophy and choroidal neovascularization imaged by FA were depicted by PV-OCT. Regions of capillary nonperfusion from diabetic retinopathy were shown by both imaging techniques; there was not complete correspondence between microaneurysms shown on FA and PV-OCT images.ConclusionsPhase-variance OCT yields high-resolution imaging of the retinal and choroidal microvasculature that compares favorably with FA.

Detailed visualization of microvascular changes in the human retina is clinically limited by the capabilities of angiography imaging, a 2D fundus photograph that requires an intravenous injection of fluorescent dye. Whereas current angiography methods enable visualization of some retinal capillary detail, they do not adequately reveal the choriocapillaris or other microvascular features beneath the retina. We have developed a noninvasive microvascular imaging technique called phase-variance optical coherence tomography (pvOCT), which identifies vasculature three dimensionally through analysis of data acquired with OCT systems. The pvOCT imaging method is not only capable of generating capillary perfusion maps for the retina, but it can also use the 3D capabilities to segment the data in depth to isolate vasculature in different layers of the retina and choroid. This paper demonstrates some of the capabilities of pvOCT imaging of the anterior layers of choroidal vasculature of a healthy normal eye as well as of eyes with geographic atrophy (GA) secondary to age-related macular degeneration. The pvOCT data presented permit digital segmentation to produce 2D depth-resolved images of the retinal vasculature, the choriocapillaris, and the vessels in Sattler's and Haller's layers. Comparisons are presented between en face projections of pvOCT data within the superficial choroid and clinical angiography images for regions of GA. Abnormalities and vascular dropout observed within the choriocapillaris for pvOCT are compared with regional GA progression. The capability of pvOCT imaging of the microvasculature of the choriocapillaris and the anterior choroidal vasculature has the potential to become a unique tool to evaluate therapies and understand the underlying mechanisms of age-related macular degeneration progression.

We present in vivo noninvasive retinal and choroidal perfusion maps with phase-variance optical coherence tomography (pvOCT). We acquired a pvOCT volumetric data set of a normal subject and visualized blood circulation in the retina and the choroid. En face projection views of the retina as well as the choroid were generated from a manually segmented volumetric data set. In addition, the processed pvOCT images were compared to current standard imaging modalities used for retinal and choroidal vasculature visualization in clinical settings, including fluorescein angiography (FA) and indocyanine green angiography (ICGA). © 2013 Copyright SPIE.

We evaluate methods to visualize human retinal micro-circulation in vivo by standard intensity-based optical coherence tomography (OCT), speckle-variance optical coherence tomography (svOCT), and phase-variance optical coherence tomography (pvOCT). En face projection views created from the same volumetric data set of the human retina using all three data processing methods are created and compared. Additionally we used support vector machine (SVM) based semi-automatic segmentation to generate en face projection views of individual retinal layers. The layers include: first, the whole inner retina (from the nerve fiber layer to the outer nuclear layer), and second, from the ganglion cell layer to the outer nuclear layer. Finally, we compare the retinal vasculature images processed from the three OCT techniques and fluorescein angiography (FA). © 2012 SPIE.

PurposeTo demonstrate the application of phase-variance optical coherence tomography (pvOCT) for contrast agent-free in vivo imaging of volumetric retinal microcirculation in the human foveal region and for extraction of foveal avascular zone dimensions.MethodsA custom-built, high-speed Fourier-domain OCT retinal imaging system was used to image retinas of two healthy subjects and eight diabetic patients. Through the acquisition of multiple B-scans for each scan location, phase differences between consecutive scans were extracted and used for phase-variance contrast, identifying motion signals from within blood vessels and capillaries. The en face projection view of the inner retinal layers segmented out from volumetric pvOCT data sets allowed visualization of a perfusion network with the foveal avascular zone (FAZ). In addition, the authors presented 2D retinal perfusion maps with pseudo color-coded depth positions of capillaries.ResultsRetinal vascular imaging with pvOCT provides accurate measurements of the FAZ area and its morphology and a volumetric perfusion map of microcapillaries. In this study using two images from each fundus fluorescein angiography (FA) and pvOCT, the measured average areas of the FAZ from two healthy subjects were below 0.22 mm(2), and each of eight diabetic patients had an enlarged FAZ area, larger than 0.22 mm(2). Moreover, the FAZ areas demonstrated a significant correlation (r = 0.91) between measurements from FA and pvOCT.ConclusionsThe high-speed pvOCT allows contrast agent-free visualization of capillary networks in the human foveal region that is analogous to fundus FA imaging. This could allow for noninvasive diagnosis and progression monitoring of diabetic retinopathy in clinical settings.

We present in vivo volumetric images of human retinal micro-circulation using Fourier-domain optical coherence tomography (Fd-OCT) with the phase-variance based motion contrast method. Currently fundus fluorescein angiography (FA) is the standard technique in clinical settings for visualizing blood circulation of the retina. High contrast imaging of retinal vasculature is achieved by injection of a fluorescein dye into the systemic circulation. We previously reported phase-variance optical coherence tomography (pvOCT) as an alternative and non-invasive technique to image human retinal capillaries. In contrast to FA, pvOCT allows not only noninvasive visualization of a two-dimensional retinal perfusion map but also volumetric morphology of retinal microvasculature with high sensitivity. In this paper we report high-speed acquisition at 125 kHz A-scans with pvOCT to reduce motion artifacts and increase the scanning area when compared with previous reports. Two scanning schemes with different sampling densities and scanning areas are evaluated to find optimal parameters for high acquisition speed in vivo imaging. In order to evaluate this technique, we compare pvOCT capillary imaging at 3x3 mm(2) and 1.5x1.5 mm(2) with fundus FA for a normal human subject. Additionally, a volumetric view of retinal capillaries and a stitched image acquired with ten 3x3 mm(2) pvOCT sub-volumes are presented. Visualization of retinal vasculature with pvOCT has potential for diagnosis of retinal vascular diseases.

We present high-speed Fourier-domain optical coherence tomography (Fd-OCT) with the phase variance based motion contrast method for visualizing retinal micro-circulation in vivo. This technique allows non-invasive visualization of a two-dimensional retinal perfusion map and concurrent volumetric morphology of retinal microvasculature with high sensitivity. The high-speed acquisition rate at 125kHz A-scans enables reduction of motion artifacts with increased scanning area if compared to previously reported results. Several scanning schemes with different sampling densities and scanning areas are evaluated to find optimal parameters for in vivo imaging. In order to evaluate this technique, we compare OCT micro-capillary imaging using the phase variance technique with fundus fluorescein angiography (FA). Additionally, volumetric visualization of blood flow for a normal subject is presented. © 2011 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).

Phase variance-based motion contrast imaging is demonstrated using a spectral domain optical coherence tomography system for the in vivo human retina. This contrast technique spatially identifies locations of motion within the retina primarily associated with vasculature. Histogram-based noise analysis of the motion contrast images was used to reduce the motion noise created by transverse eye motion. En face summation images created from the 3D motion contrast data are presented with segmentation of selected retinal layers to provide non-invasive vascular visualization comparable to currently used invasive angiographic imaging. This motion contrast technique has demonstrated the ability to visualize resolution-limited vasculature independent of vessel orientation and flow velocity.

The walls of conduit arteries undergo cyclic stretching from the periodic fluctuation of arterial pressure. Atherosclerotic lesions have been shown to localize to regions of excessive stretching of the arterial wall. We employed a displacement encoding with stimulated echoes (DENSE) sequence to image the motion of the common carotid artery wall and map the two-dimensional (2D) circumferential strain. The sequence utilizes a fully-balanced steady-state free-precession (SSFP) readout with 0.60 mm in-plane resolution. Preliminary results in volunteers at 1.5T (N = 4) and 3.0T (N = 17) are compared to measurements of the lumen circumference from cine images. The agreement between the two independent measurements at both field strengths (P < or = 0.001) supports the use of DENSE as a means to map the pulsatile strain in the carotid artery wall.

The metameric organization of the vertebrate hindbrain into rhombomeres appears to result from the patterned expression of several transcription factors and putative signaling molecules. We have applied a refined single-cell reverse transcription-polymerase chain reaction strategy to examine the molecular logic proposed to pattern the hindbrain at the single-cell level. This technique allows analysis of the concurrent expression of several genes within an individual cell at higher sensitivity than by in situ hybridization. Our results demonstrate that cells in rhombomere (r) 4 and r5 are heterogeneous in their expression of Hoxa-3, Hoxb-2, Sek-1, and Krox-20, suggesting that single cells are dynamically regulating their rhombomere-specific gene-expression profiles. Furthermore, the strong correlation between Sek-1 and Krox-20 expression at stage 12 was greatly diminished by stage 16, suggesting that the proposed interdependence of these two genes is present only at early stages of hindbrain development.

Background: Human Apolipoprotein (APOE) has three isoforms, ε2, ε3, and ε4 among which ε4 (APOE4) confers the highest risk for late‐onset Alzheimer's disease (AD). APOE4 is also the most prone to aggregate among APOE isoforms. Current evidence strongly suggests that APOE aggregation leads to neuronal dysfunction and eventually to AD. APOE4 increases amyloid plaques and neurofibrillary tangles and decreases synapses and neuronal survival. These phenotypes are alleviated by decreasing APOE4 aggregation. Method: We analyzed APOE aggregation using fluorescence lifetime imaging microscopy (FLIM) in combination with Forster resonance energy transfer (FRET). APOE isoforms tagged with E2GFP or mRuby2 were within 10 nm or less, i.e. in an aggregate the fluorescence lifetime of the donor was shortened and signal intensity was diminished which was monitored using FLIM‐FRET and analyzed using the Phasor approach. APOE aggregation was also confirmed by using small‐angle X‐ray scattering (SAXS) of the Sarkosyl extracts of the cells. Secreted APOE‐Tdtomato was isolated from BHK cell media and analyzed by ion mobility assay. Human neurons were incubated with APOE‐Tdtomato isolated from the media. Results: APOE4 aggregated more than APOE3 in living BHK cells as shown via FLIM‐FRET microscopy. APOE4 aggregated less when its lipidation was induced by ABCA1 expression in line with data published by us and others. These results were confirmed via SAXS. APOE was secreted in HDL particles from BHK cells both in the presence or absence of ABCA1 while ABCA1 increased secretion for both APOE3 and APOE4. Finally, tagged‐APOE secreted to the media was endocytosed by human neurons. Conclusions: FLIM‐FRET is a feasible method to analyze APOE aggregation in living cells. Isolated APOE can be used to study APOE endocytosis in human neurons. [ABSTRACT FROM AUTHOR]