Bundle Tip Research Articles

Ptycho-endoscopy on a lensless ultrathin fiber bundle tip

Synthetic aperture radar (SAR) utilizes an aircraft-carried antenna to emit electromagnetic pulses and detect the returning echoes. As the aircraft travels across a designated area, it synthesizes a large virtual aperture to improve image resolution. Inspired by SAR, we introduce synthetic aperture ptycho-endoscopy (SAPE) for micro-endoscopic imaging beyond the diffraction limit. SAPE operates by hand-holding a lensless fiber bundle tip to record coherent diffraction patterns from specimens. The fiber cores at the distal tip modulate the diffracted wavefield within a confined area, emulating the role of the ‘airborne antenna’ in SAR. The handheld operation introduces positional shifts to the tip, analogous to the aircraft’s movement. These shifts facilitate the acquisition of a ptychogram and synthesize a large virtual aperture extending beyond the bundle’s physical limit. We mitigate the influences of hand motion and fiber bending through a low-rank spatiotemporal decomposition of the bundle’s modulation profile. Our tests demonstrate the ability to resolve a 548-nm linewidth on a resolution target. The achieved space-bandwidth product is ~1.1 million effective pixels, representing a 36-fold increase compared to that of the original fiber bundle. Furthermore, SAPE’s refocusing capability enables imaging over an extended depth of field exceeding 2 cm. The aperture synthesizing process in SAPE surpasses the diffraction limit set by the probe’s maximum collection angle, opening new opportunities for both fiber-based and distal-chip endoscopy in applications such as medical diagnostics and industrial inspection.

Inferring entropy production rate from partially observed Langevin dynamics under coarse-graining.

The entropy production rate (EPR) measures time-irreversibility in systems operating far from equilibrium. The challenge in estimating the EPR for a continuous variable system is the finite spatiotemporal resolution and the limited accessibility to all of the nonequilibrium degrees of freedom. Here, we estimate the irreversibility in partially observed systems following oscillatory dynamics governed by coupled overdamped Langevin equations. We coarse-grain an observed variable of a nonequilibrium driven system into a few discrete states and estimate a lower bound on the total EPR. As a model system, we use hair-cell bundle oscillations driven by molecular motors, such that the bundle tip position is observed, but the positions of the motors are hidden. In the observed variable space, the underlying driven process exhibits second-order semi-Markov statistics. The waiting time distributions (WTD), associated with transitions among the coarse-grained states, are non-exponential and convey the information on the broken time-reversal symmetry. By invoking the underlying time-irreversibility, we calculate a lower bound on the total EPR from the Kullback–Leibler divergence (KLD) between WTD. We show that the mean dwell-time asymmetry factor – the ratio between the mean dwell-times along the forward direction and the backward direction, can qualitatively measure the degree of broken time reversal symmetry and increases with finer spatial resolution. Finally, we apply our methodology to a continuous-time discrete Markov chain model, coarse-grained into a linear system exhibiting second-order semi-Markovian statistics, and demonstrate the estimation of a lower bound on the total EPR from irreversibility manifested only in the WTD.

All-fiber-optic LIBS system for tissue differentiation: A prospect for endoscopic smart laser osteotomy

Minimally-invasive laser surgeries could benefit from a fiber-optic laser-induced breakdown spectroscopy (FO-LIBS) setup for real-time tissue characterization. In FO-LIBS, the sample receives limited light irradiance due to the fiber's low damage threshold and diminished laser beam quality. Therefore, the plasma created with FO-LIBS is less luminant than that of free-space LIBS. Furthermore, only a small portion of plasma emission can be collected, as the lens's size at the fiber tip is restricted to fit inside the narrow channel of an endoscope. A high optical throughput Echelle spectrometer was developed to compensate for low-intensity light collection with FO-LIBS. The Echelle spectrometer was tested for tissue differentiation when combined with a flexible fiber bundle delivery setup and a small lens at the bundle's tip. The customized FO-LIBS setup, coupled with multivariate data analysis, successfully differentiated bone from surrounding soft tissue (muscle, fat, and bone marrow) with 100% cross-validated (CV) sensitivity and specificity. The CV sensitivity and specificity for differentiation between all tissues were 90.2% and 96.7%, respectively. The results demonstrate, to the best of our knowledge, the first flexible FO-LIBS system, which may provide a further step towards the development of a smart endoscopic laser scalpel.

Diffused light attenuation at 664 nm for PDT in salted cadaver brain.

We investigated light attenuation at 664 nm, which is the excitation wavelength of photodynamic therapy (PDT) using talaporfin sodium, in a salted cadaver brain. Estimation of therapeutic lesions is important to ensure the effectiveness and safety of brain tumor PDT. Previously reported optical properties of the human brain vary widely. In this study, we measured the light attenuation in brain tissue using a practical method. We employed a salted cadaver brain, in which the mechanical and optical properties can be maintained as close as possible to those under operative conditions. A neuroendoscope was inserted into the brain until the cerebral ventricle was reached. A thin cylindrical diffuser probe was advanced 10 mm from the endoscope tip. By another path from the brain surface, an optical fiber for measurement was inserted into a puncture needle, and a pair of needles was used to puncture the tissue and reach the same cerebral ventricle in which the endoscope tip was positioned. The attenuation of light intensities in the frontal lobe and cerebellum was measured by varying the bundle tip position. The starting positions of the bundle were confirmed by the endoscopic view. The measured light intensity attenuations were fitted with an exponential curve. The following attenuation coefficients were obtained: 0.20 ± 0.05 mm-1 in the cerebrum and 0.27 ± 0.05 mm-1 in the cerebellum. As conventional spectroscopic measurement may overestimate attenuation in the whole tissue, in situ measurement using the withdrawal technique might be appropriate for measurement of inhomogeneous biological tissues.

Generation of mixed-mode stochastic oscillations in a hair bundle model

We consider a mathematical model of spontaneous mechanical oscillations of hair bundles in bullfrog saccular hair cells. This two-dimensional model describes dynamics of the positions of the bundle tip and molecular motors. The main control parameters of this model are the calcium feedback strength and the maximal force of molecular motors. The dynamics of hair bundles is subject to the random disturbances. We study noise-induced oscillations in the parametric zone where the equilibria are the only attractors of the system. We analyze the probabilistic mechanism of the generation of mixed-mode stochastic oscillations with the help of the stochastic sensitivity functions and confidence domains. The constructive abilities of this mathematical technique in the analysis of the stochastic effects in the hair bundle model are demonstrated.

Auditory cortex interneuron development requires cadherins operating hair-cell mechanoelectrical transduction

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15- and Cdhr23-expressing interneuron precursors in Cdhr23-/- and Cdhr15-/- mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23- and Cdhr15-expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an "early adhesion code" targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients.

A nano-microstructured artificial-hair-cell-type sensor based on topologically graded 3D carbon nanotube bundles

A design for a unique artificial-hair-cell-type sensor (AHCTS) based entirely on 3D-structured, vertically aligned carbon nanotube (CNT) bundles is introduced. Standard microfabrication techniques were used for the straightforward micro-nano integration of vertically aligned carbon nanotube arrays composed of low-layer multi-walled CNTs (two to six layers). The mechanical properties of the carbon nanotube bundles were intensively characterized with regard to various substrates and CNT morphology, e.g. bundle height. The CNT bundles display excellent flexibility and mechanical stability for lateral bending, showing high tear resistance. The integrated 3D CNT sensor can detect three-dimensional forces using the deflection or compression of a central CNT bundle which changes the contact resistance to the shorter neighboring bundles. The complete sensor system can be fabricated using a single chemical vapor deposition (CVD) process step. Moreover, sophisticated external contacts to the surroundings are not necessary for signal detection. No additional sensors or external bias for signal detection are required. This simplifies the miniaturization and the integration of these nanostructures for future microsystem set-ups. The new nanostructured sensor system exhibits an average sensitivity of 2100 ppm in the linear regime with the relative resistance change per micron (ppm μm−1) of the individual CNT bundle tip deflection. Furthermore, experiments have shown highly sensitive piezoresistive behavior with an electrical resistance decrease of up to ∼11% at 50 μm mechanical deflection. The detection sensitivity is as low as 1 μm of deflection, and thus highly comparable with the tactile hair sensors of insects, having typical thresholds on the order of 30–50 μm. The AHCTS can easily be adapted and applied as a flow, tactile or acceleration sensor as well as a vibration sensor. Potential applications of the latter might come up in artificial cochlear systems. In particular, the stable mechanical bending of the sensor up to 90° opens up unique application opportunities.

Real-Time Tissue Differentiation Using Fiber Optic Sensing in Laser Catheters1

Real-Time Tissue Differentiation Using Fiber Optic Sensing in Laser Catheters1

Sound Transduction in the Mammalian Outer Hair Cells: Prestin Activity is Required for Proper Deflection of the Stereocilia Bundle

The outer hair cell (OHC) body is capable of prestin-driven electromotility leading to force generation that increases the vibration of the hearing organ critical for auditory sensitivity. At the cell's apex, the stereocilia bundle deflects as a unit during sound stimulation (Fridberger et al, 2006). Such a deflection converts nanometric displacements into electrical signals transmitted to the auditory nerve. Very little is however known about how sound stimuli cause the bundle to deflect, especially, the possible contribution of prestin-induced cell body vibrations to this deflection has never been investigated. Here we investigated the influence of the membrane protein prestin activity on the bundle deflection, in an intact ear preparation from the Guinea pig. Prestin was previously shown to be specifically inactivated by salicylate and tributyltin. Using an approach combining rapid confocal imaging and optical flow-based computation, the bundle deflection was studied under simultaneous sound stimulus administered at 50-350HZ, a frequency band typical of OHCs vibrations in the apex of the cochlea. To our surprise and irrespective of the prestin inhibitor used, sound-induced bundle deflection drastically increases, specifically, near the best frequency whose position was altered. Likewise, the vibration of the bundle tip intensified. Moreover, the shape of the bundle deflection's pattern was affected. Our data challenge the general assumption that prestin inactivation decreases the vibrations of the cochlear's structures. Because no consistent change was observed for vibrations of the reticular lamina, the increase in the bundle deflection may be caused by a robust vibration of the top. The data suggest that prestin motor's activity regulates the tuning of the bundle vibrations and may explain how the stereociliary and saumatic amplifiers interact during sound transduction in the mammalian ear.

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length-dependent increase in the catastrophe frequency that is mediated by kinesin-8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin-8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.

A Virtual Hair Cell, I: Addition of Gating Spring Theory into a 3-D Bundle Mechanical Model

We have developed a virtual hair cell that simulates hair cell mechanoelectrical transduction in the turtle utricle. This study combines a full three-dimensional hair bundle mechanical model with a gating spring theory. Previous mathematical models represent the hair bundle with a single degree of freedom system which, we have argued, cannot fully explain hair bundle mechanics. In our computer model, the tip link tension and fast adaptation modulator kinetics determine the opening and closing of each channel independently. We observed the response of individual transduction channels with our presented model. The simulated results showed three features of hair cells in vitro. First, a transient rebound of the bundle tip appeared when fast adaptation dominated the dynamics. Second, the dynamic stiffness of the bundle was minimized when the response-displacement ( I- X) curve was steepest. Third, the hair cell showed “polarity”, i.e., activation decreased from a peak to zero as the forcing direction rotated from the excitatory to the inhibitory direction.

Short-term retention of actin filament binding proteins on lamellipodial actin bundles

Actin filaments are organised into sub-compartments of meshwork and bundles in lamellipodia. Localisation of fascin, the LIM and SH3 domain protein 1 (lasp-1), and lasp-2 to the bundles suggest their involvement in that organisation; however, their contributions remain unclear. We have compared the turnover of these proteins with actin at the bundle. After photobleaching, EGFP-actin recovered inwards from the bundle tip, consistent with the retrograde flow by treadmilling. In contrast, the recovery of EGFP-fascin, -lasp-1 and -lasp-2 occurred from the anterograde direction. These results suggest that these molecules would participate in the stabilisation of bundles but not in initiation.

Observation of Coulomb Blockade and Interference Using a Carbon Nanotube Tip

Using a carbon nanotube (CNT) bundle tip as a field emitter, we have observed both Coulomb blockade and interference fringes in consecutive measurements. When Coulomb blockade is observed, decoherence of the electron wave occurs. This was observed using a double-tunnel junction consisting of a CNT tip and a CNT sample. When the CNT sample was projected onto a screen by an electron wave from the CNT tip, interference fringes were observed. This shows that the electron is first observed at the central electrode as a Coulomb blockade, next it is observed at the CNT sample as current when the electron leaves from the central electrode (the CNT tip) and finally observed as fringes passing through the CNT sample. These results show that the system's wave function cannot be written as a direct product of independent substates.

Tip-link integrity on chick tall hair cell stereocilia following intense sound exposure

Hair bundle tip links have been implicated in the process of hair cell transduction, and previous studies have shown that acoustic overstimulation or exposure to low calcium can disrupt them. Severed tip links would thus be expected to cause a loss in hair cell function. This study investigates the presence of tip links on chick tall hair cells at three exposure durations and three recovery durations. After 4, 24, or 48 h of exposure, and 24, 96, and 288 h of recovery, the basilar papilla was harvested and prepared for scanning electron microscopy. Photomicrographs of hair bundles from sound-exposed and age-matched control ears were obtained in regions of the papilla adjacent to the ‘patch’ lesion. The percentage of tip links present on these hair bundles was determined from the photomicrographs. After 4, 24, or 48 h of exposure, an average of 49%, 41.1% and 52% of the observed sensory hairs exhibited links. This was significantly lower than that seen in the control ears (71.2%). There also was a reliable recovery of tip links between 24 and 48 h of exposure. The recovery continued and by 24 h post exposure, tip links were present on 61.3% of the sensory hairs. At subsequent recovery intervals, the mean number of tip links on sound-exposed tall hair cells was statistically the same as seen on control cells. The results indicated a predictable loss in the number of tip links during the exposure and their restoration within a relatively short time after the exposure. This structural damage to the tall hair cell, and its recovery, could account for some of the loss and recovery of function in the auditory periphery of these sound-damaged chicks.

The Ankle-Link Antigen: an Epitope Sensitive to Calcium Chelation Associated with the Hair-Cell Surface and the Calycal Processes of Photoreceptors

A monoclonal antibody, mAb E40, that specifically recognizes hair cells and photoreceptors was derived from a mouse immunized with a membrane fraction prepared from the sensory maculae of the chick inner ear. In the mature chick inner ear, punctate labeling is observed along each stereocilium, but staining is mostly concentrated around the basal end of the sensory hair bundles, where it is closely associated with surface specializations known as ankle links. The epitope recognized by mAb E40 is therefore referred to as the ankle-link antigen (ALA). During early embryogenesis, the ALA is initially distributed evenly over the surface of the hair bundle. As development proceeds, it becomes more restricted to the base of the hair bundle, although a spot of the ALA remains associated with the bundle tip until just before hatching. In the eye, mAb E40 stains the calycal processes of photoreceptors. When maculae and retinae are treated with the calcium chelator BAPTA at room temperature, the ALA disappears. BAPTA-induced loss of the ALA from the hair-bundle surface is substantially reduced by lowering the temperature to 2 degrees C. The ALA and ankle links reappear on the hair-bundle surface when cells are cultured for 20 hr after BAPTA treatment. BAPTA sensitivity and recovery after BAPTA-induced loss are properties similar to those described for the tip link, a surface structure thought to gate the mechanotransducer channel. However, unlike the tip link, the ALA and ankle links are sensitive to subtilisin treatment. The results define a new component of the hair-bundle surface, with properties both common to and distinct from those of the tip link.

Mechanical properties of sensory hair bundles are reflected in their Brownian motion measured with a laser differential interferometer.

By optically probing with a focused, low-power laser beam, we measured the spontaneous deflection fluctuations of the sensory hair bundles on frog saccular hair cells with a sensitivity of about 1 pm/square root of Hz. The preparation was illuminated by two orthogonally polarized laser beams separated by only about 0.2 microns at their foci in the structure under investigation. Slight movement of the object from one beam toward the other caused a change of the phase difference between the transmitted beams and an intensity modulation at the detector where the beams interfered. Maintenance of the health of the cells and function of the transduction mechanism were occasionally confirmed by measuring the intracellular resting potential and the sensitivity of transduction. The root-mean-square (rms) displacement of approximately 3.5 nm at a hair bundle's tip suggests a stiffness of about 350 microN/m, in agreement with measurements made with a probe attached to a bundle's tip. The spectra resemble those of overdamped harmonic oscillators with roll-off frequencies between 200 and 800 Hz. Because the roll-off frequencies depended strongly on the viscosity of the bathing medium, we conclude that hair-bundle motion is mainly damped by the surrounding fluid.