Haptic guidance for microrobotic intracellular injection

The ability for a bio-operator to utilise a haptic device to manipulate a microrobot for intracellular injection offers immense benefits. One significant benefit is for the bio-operator to receive haptic guidance while performing the injection process. In order to address this, this paper investigates the use of haptic virtual fixtures for cell injection and proposes a novel force field virtual fixture. The guidance force felt by the bio-operator is determined by force field analysis within the virtual fixture. The proposed force field virtual fixture assists the bio-operator when performing intracellular injection by limiting the micropipette tip's motion to a conical volume as well as recommending the desired path for optimal injection. A virtual fixture plane is also introduced to prevent the bio-operator from moving the micropipette tip beyond the deposition target inside the cell. Simulation results demonstrate the operation of the guidance system.

Optimal sensor separation for AoA based localization via linear sensor array

This paper investigates the linear separation requirements for Angle-of-Arrival (AoA) sensors, in order to achieve the optimal performance in estimating the position of a target from multiple and typically noisy sensor measurements. We analyse the sensor-target geometry in terms of the Cramer-Rao inequality and the corresponding Fisher information matrix, in order to characterize localization performance with respect to the linear spacial distribution. Here we consider a situation where one sensor is fixed and the rest are free to be positioned in a linear array.

Haptic microrobotic intracellular injection assistance using virtual fixtures

In manual cell injection the operator relies completely on visual information for task feedback and is subject to extended training times as well as poor success rates and repeatability. From this perspective, enhancing human-in-the-loop intracellular injection through haptic interaction offers significant benefits. This paper outlines two haptic virtual fixtures aiming to assist the human operator while performing cell injection. The first haptic virtual fixture is a parabolic force field designed to assist the operator in guiding the micropipette's tip to a desired penetration point on the cell's surface. The second is a planar virtual fixture which attempts to assist the operator from moving the micropipette's tip beyond the deposition target location inside the cell. Preliminary results demonstrate the operation of the haptically assisted microrobotic cell injection system.

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies.

Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen : intracellular targeting to the amyloplast

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

Secure localization with attack detection in wireless sensor networks

Rapid technological advances have enabled the development of low-cost sensor networks for various monitoring tasks, where it is important to estimate the positions of a number of regular sensor nodes whose locations cannot be known apriori. We address the problem of localizing the regular nodes with range-based location references obtained from certain anchor nodes referred to as beacons, particularly in an adverse environment where some of the beacons may be compromised. We propose an innovative modular solution featuring two lightweight modules that are for dedicated functionalities, respectively, but can also be closely integrated. First, we harness simple geometric triangular rules and an efficient voting technique to enable the attack detection module, which identifies and filters out malicious location references. We then develop a secure localization module that computes and clusters certain reference points, and the position of the concerned regular node is estimated with the centroid of the most valuable reference points identified. Extensive simulations show that our attack detection module can detect compromised beacons effectively, and the secure localization module can subsequently provide a dependable localization service in terms of bounded estimation error. The integrated system turns out to be tolerant of malicious attacks even in highly challenging scenarios.

A robust solution to the stereo-vision-based simultaneous localization and mapping problem with steady and moving landmarks

The problem of visual simultaneous localization and mapping (SLAM) is examined in this paper using recently developed ideas and algorithms from modern robust control and estimation theory. A nonlinear model for a stereo-vision-based sensor is derived that leads to nonlinear measurements of the landmark coordinates along with optical flow-based measurements of the relative robot-landmark velocity. Using a novel analytical measurement transformation, the nonlinear SLAM problem is converted into the linear domain and solved using a robust linear filter. Actually, the linear filter is guaranteed stable and the SLAM state estimation error is bounded within an ellipsoidal set. A mathematically rigorous stability proof is given that holds true even when the landmarks move in accordance with an unknown control input. No similar results are available for the commonly employed extended Kalman filter, which is known to exhibit divergence and inconsistency characteristics in practice. A number of illustrative examples are given using both simulated and real vision data that further validate the proposed method.

A critical role for the short intracellular C terminus in receptor activity-modifying protein function

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Gαs, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.