pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters

Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscopy's dipole spread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Green's tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 1.14 degree orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration, in 6 dimensions.

Structural Diversity of Photoswitchable Sphingolipids for Optodynamic Control of Lipid Raft Microdomains

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other saturated lipids and cholesterol into lipid rafts; liquid-ordered (Lo) microdomains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral localization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains, to develop a set of photoswitchable sphingolipids, with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran (THP)-blocked sphingosine), able to shuttle between liquid-ordered (Lo) and liquid-disordered (Ld) regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photo-isomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that all sphingosine-(Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photolipids behave similarly, promoting a reduction in Lo domain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having THP groups that block H-bonding at the sphingosine backbone (Azo-THP-SM, Azo-THP-Cer) induce an increase in the Lo domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable Lo lipid raft domains.

WW, PH and C-Terminal Domains Cooperate to Direct the Subcellular Localizations of PLEKHA5, PLEKHA6 and PLEKHA7

PLEKHA5, PLEKHA6, and PLEKHA7 (WW-PLEKHAs) are members of the PLEKHA family of proteins that interact with PDZD11 through their tandem WW domains. WW-PLEKHAs contribute to the trafficking and retention of transmembrane proteins, including nectins, Tspan33, and the copper pump ATP7A, at cell-cell junctions and lateral membranes. However, the structural basis for the distinct subcellular localizations of PLEKHA5, PLEKHA6, and PLEKHA7 is not clear. Here we expressed mutant and chimeric proteins of WW-PLEKHAs in cultured cells to clarify the role of their structural domains in their localization. We found that the WW-mediated interaction between PLEKHA5 and PDZD11 is required for their respective association with cytoplasmic microtubules. The PH domain of PLEKHA5 is required for its localization along the lateral plasma membrane and promotes the lateral localization of PLEKHA7 in a chimeric molecule. Although the PH domain of PLEKHA7 is not required for its localization at the adherens junctions (AJ), it promotes a AJ localization of chimeric proteins. The C-terminal region of PLEKHA6 and PLEKHA7 and the coiled-coil region of PLEKHA7 promote their localization at AJ of epithelial cells. These observations indicate that the localizations of WW-PLEKHAs at specific subcellular sites, where they recruit PDZD11, are the result of multiple cooperative protein-lipid and protein-protein interactions and provide a rational basis for the identification of additional proteins involved in trafficking and sorting of WW-PLEKHAs.

Mono-Vision Based Lateral Localization System of Low-Cost Autonomous Vehicles Using Deep Learning Curb Detection

The localization system of low-cost autonomous vehicles such as autonomous sweeper requires a highly lateral localization accuracy as the vehicle needs to keep a near lateral-distance between the side brush system and the road curb. Existing methods usually rely on a global navigation satellite system that often loses signal in a cluttered environment such as sweeping streets between high buildings and trees. In a GPS-denied environment, map-based methods are often used such as visual and LiDAR odometry systems. Apart from heavy computation costs from feature extractions, they are too expensive to meet the low-price market of the low-cost autonomous vehicles. To address these issues, we propose a mono-vision based lateral localization system of an autonomous sweeper. Our system relies on a fish-eye camera and precisely detects road curbs with a deep curb detection network. Curbs locations are then referred to as straightforward marks to control the lateral motion of the vehicle. With our self-recorded dataset, our curb detection network achieves 93% pixel-level precision. In addition, experiments are performed with an intelligent sweeper to prove the accuracy and robustness of our proposed approach. Results demonstrate that the average lateral distance error and the maximum invalid rate are within 0.035 m and 9.2%, respectively.

Localization of Deep Voids through Geophysical Signatures of Secondary Dewatering Features

Discrete deep targets are a significant challenge for most surface-based geophysical techniques, even when considering high property contrasts. Generally, surface-based geophysical methods lose lateral and vertical resolution with depth as a result of poor measurement geometry and increased signal attenuation. The poor measurement geometry can be overcome through use of cross-borehole methods, but lateral localization is still needed for optimal borehole placement. As such, a relatively small, deep void located near the maximum depth of investigation is very unlikely to be detected. Yet, secondary features associated with these voids can be exploited for enhanced detection performance. When voids are located below the groundwater table a significant amount of dewatering and pumping is required to make them a functional passageway. This dewatering not only removes water from the void space but also the surrounding formation, resulting in a much larger, if more diffuse, secondary target: an induced groundwater table gradient. Many geophysical sensing methods are sensitive to subsurface moisture content. Here we implement a two-dimensional (2D) joint-petrophysical mixing-model, using inverted electrical resistivity tomography and inverted seismic refraction models to sense changes in the groundwater table gradient. Results are validated using depth to bedrock, groundwater-surface water information, ground-penetrating radar, and time-domain reflectometry methods. Our initial proof of concept is applied to a shallow area with a significant soil moisture gradient, through different surface soil types and bedrock. The 2D joint-petrophysical mixing-model results were used to generate an estimate of air, moisture, and matrix percent fractions in the investigation area, providing a clear delineation of the groundwater surface.

Examination of the relation of localization of placenta at 18-24 weeks of gestation by ultrasonography with the development of preeclampsia later in pregnancy

Background: The aim of the study was to examine the relation of localization of placenta at 18-24 weeks of gestation by ultrasonography with the development of preeclampsia later in pregnancy.Methods: Hospital based Prospective observational Cohort study at department of Obstetrics and Gynecology, Dr. BSA Hospital Delhi. 150 pregnant women of gestational age18-24 weeks attending ANC clinic were enrolled for ultrasound examination and on the basis of ultrasonography (USG) findings placenta was classified as Group-A (patient found to have laterally located placenta). And Group-B (patients found to have centrally located placenta). All women in both the group were followed up regularly till term. Study duration was 1 year, June 2017-June 2018.Results: The overall risk of developing Preeclampsia with a late rally located placenta was 8.5(odds ratio) with 95% confidence interval (4.0339 to17.9108). This difference was highly statistically significant (p<.0001). Placental laterality has a sensitivity of 77.27%, beside that it has a good specificity of 71.43% and negative predictive value of 80%. Lateral localization of placenta by ultrasonography at mid trimester can be used as a screening test. The presence of urine albumin in group A was found in 70.67% as compared to 13.33% in group B. This difference was statistically significant. (p=0.0001). Around 72% of total complications were seen in Group-A as compared to 28% in Group-B. This difference was found to be statistically significant (p=0.028).Conclusions: Significant correlation exists between placental laterality and the development of preeclampsia and thus placental localization by ultrasonography in midtrimester (at 18-24 weeks) can be used for prediction of development of preeclampsia later in pregnancy.

Nanometric axial localization of single fluorescent molecules with modulated excitation

Strategies have been developed in LIDAR to perform distance measurements for non-coherent emission in sparse samples based on excitation modulation. Super-resolution fluorescence microscopy is also striving to perform axial localization but through entirely different approaches. Here we revisit the amplitude modulated LIDAR approach to reach nanometric localization precision and we successfully adapt it to bring distinct advantages to super-resolution microscopy. The excitation pattern is performed by interference enabling the decoupling between spatial and time modulation. The localization of a single emitter is performed by measuring the relative phase of its linear fluorescent response to the known shifting excitation field. Taking advantage of a tilted interfering configuration, we obtain a typical axial localization precision of 7.5 nm over the entire field of view and the axial capture range, without compromising on the acquisition time, the emitter density or the lateral localization precision. The interfering pattern being robust to optical aberrations, this modulated localization (ModLoc) strategy is particularly well suited for observations deep in the samples. Images performed on various biological samples show that the localization precision remains nearly constant up to several micrometers.

No effect of triple-pulse TMS medial to intraparietal sulcus on online correction for target perturbations during goal-directed hand and foot reaches

Posterior parietal cortex (PPC) is central to sensorimotor processing for goal-directed hand and foot movements. Yet, the specific role of PPC subregions in these functions is not clear. Previous human neuroimaging and transcranial magnetic stimulation (TMS) work has suggested that PPC lateral to the intraparietal sulcus (IPS) is involved in directing the arm, shaping the hand, and correcting both finger-shaping and hand trajectory during movement. The lateral localization of these functions agrees with the comparably lateral position of the hand and fingers within the motor and somatosensory homunculi along the central sulcus; this might suggest that, in analogy, (goal-directed) foot movements would be mediated by medial portions of PPC. However, foot movement planning activates similar regions for both hand and foot movement along the caudal-to-rostral axis of PPC, with some effector-specificity evident only rostrally, near the central regions of sensorimotor cortex. Here, we attempted to test the causal involvement of PPC regions medial to IPS in hand and foot reaching as well as online correction evoked by target displacement. Participants made hand and foot reaches towards identical visual targets. Sometimes, the target changed position 100-117 ms into the movement. We disturbed cortical processing over four positions medial to IPS with three pulses of TMS separated by 40 ms, both during trials with and without target displacement. We timed TMS to disrupt reach execution and online correction. TMS did not affect endpoint error, endpoint variability, or reach trajectories for hand or foot. While these negative results await replication with different TMS timing and parameters, we conclude that regions medial to IPS are involved in planning, rather than execution and online control, of goal-directed limb movements.