Towards space-deployable laser stabilization systems based on vibration-insensitive cubic cavities with crystalline coatings.

We present the development of a transportable laser frequency stabilization system with application to both optical clocks and a next-generation gravity mission (NGGM) in space. This effort leverages a 5-cm long cubic cavity with crystalline coatings operating at room temperature and with a center wavelength of 1064 nm. The cavity is integrated in a custom vacuum chamber with dedicated low-noise locking electronics. Our vacuum-mounted cavity and control system are well suited for space applications, exhibiting state-of-the-art noise performance while being resilient to radiation exposure, vibration, shock, and temperature variations. Furthermore, we demonstrate a robust means of automatically (re)locking the laser to the cavity when resonance is lost. We show that the mounted cavity is capable of reaching technology readiness level (TRL) 6, paving the way for high-performance ultrastable laser systems and eventually optical atomic clocks amenable to future satellite platforms.

Dual-axis cubic cavity for drift-compensated multi-wavelength laser stabilisation.

We describe a 'clock control unit' based on a dual-axis cubic cavity (DACC) for the frequency stabilisation of lasers involved in a strontium optical lattice clock. The DACC, which ultimately targets deployment in space applications, provides a short-term stable reference for all auxiliary lasers-i.e. cooling, clear-out, and optical lattice-in a single multi-band cavity. Long-term cavity drift is compensated by a feed-forward scheme exploiting a fixed physical relation to an orthogonal second cavity axis; either by reference to an ultrastable 698 nm clock laser, or by exploiting the differential drift between orthogonal axes extracted by a single laser in common view. Via a change of mirror set in the cavity axis accessed by the clock laser, the system could also provide stabilisation for sub-Hz linewidths at the 698 nm clock wavelength, fulfilling all stabilisation requirements of the clock.

A novel micropit device integrates automated cell positioning by dielectrophoresis and nuclear transfer by electrofusion.

Nuclear transfer (NT) cloning involves manual positioning of individual donor-recipient cell couplets for electrofusion. This is time-consuming and introduces operator-dependent variation as a confounding parameter in cloning trials. In order to automate the NT procedure, we developed a micro-fluidic device that integrates automated cell positioning and electrofusion of isolated cell couplets. A simple two layer micro-fluidic device was fabricated. Thin film interdigitated titanium electrodes (300 nm thick, 250 microm wide and 250 microm apart) were deposited on a solid borosilicate glass substrate. They were coated with a film of electrically insulating photosensitive epoxy polymer (SU-8) of either 4 or 22 microm thickness. Circular holes ("micropits") measuring 10, 20, 30, 40 or 80 microm in diameter were fabricated above the electrodes. The device was immersed in hypo-osmolar fusion buffer and manually loaded with somatic donor cells and recipient oocytes. Dielectrophoresis (DEP) was used to attract cells towards the micropit and form couplets on the same side of the insulating film. Fusion pulses between 80 V and 120 V were applied to each couplet and fusion scored under a stereomicroscope. Automated couplet formation between oocytes and somatic cells was achieved using DEP. Bovine oocyte-oocyte, oocyte-follicular cells and oocyte-fibroblast couplets fused with up to 69% (n = 13), 50% (n = 30) and 78% (n = 9) efficiency, respectively. Fusion rates were comparable to parallel plate or film electrodes that are conventionally used for bovine NT. This demonstrates proof-of-principle that a micropit device is capable of both rapid cell positioning and fusion.

Coplanar film electrodes facilitate bovine nuclear transfer cloning.

Automated lab on chip systems offer increased throughput and reproducibility, but the implementation of microelectrodes presently relies on miniaturization of parallel plate electrodes that are time consuming and costly to fabricate. Electric field modelling of open electrofusion chambers suggested that widely spaced (> or =2 mm) coplanar film electrodes should result in similar cell fusion rates as parallel plate electrodes provided the cell positioning was roughly midway between the electrodes. This hypothesis was investigated by electrofusion trials of bovine oocyte-donor cell couplets used in nuclear transfer (NT) cloning. Comparative experiments with reference parallel plate electrodes were conducted as controls. Coplanar fusion rates > or = 90% were demonstrated for embryonic blastomeres, follicular cells and fetal and adult fibroblasts as NT donor cells. For embryonic and adult cell types, there was no significant difference in fusion rate between coplanar and parallel plate electrodes. For both electrode geometries, fusion efficiency with adult fibroblasts was highest at a calculated field strength of 2.33 kV/cm. The coplanar electrodes required a voltage pi/2 times greater than parallel plate electrodes to achieve equivalent field strength when the couplets are placed midway between the electrodes.

Electroporation of Ishikawa cells: analysis by flow cytometry.

Electroporation facilitates loading of cells with molecules and substances that are normally membrane impermeable. Flow cytometry is used in this study to examine the effects of the application of electroporation-level monopolar electric field pulses of varying electrical field strength on Ishikawa endometrial adenocarcinoma cells. Analysis of the fluorescence versus forward scatter plots corroborates the well-recognised threshold and cell size dependence characteristics of electroporation, but also shows the progression of cell lysis and generation of particulate material. Two 500 µs monopolar rectangular pulses ranging from 1.0 × 105 to 2.5 × 105 V/m were used to electroporate the cells. Electroporation yields (fraction of viable cells exhibiting significant propidium iodide uptake) ranged from 0 to 97%, with viability ranging between 78 and 34% over the electric field strength range tested. The higher electric field strength pulses not only reduced cell viability, but also generated a substantial amount of sub-cellular sized particulate material indicating cells have been physically disrupted enough to create these particles.

AC electric field induced dipole-based on-chip 3D cell rotation.

The precise rotation of suspended cells is one of the many fundamental manipulations used in a wide range of biotechnological applications such as cell injection and enucleation in nuclear transfer (NT) cloning. Noticeably scarce among the existing rotation techniques is the three-dimensional (3D) rotation of cells on a single chip. Here we present an alternating current (ac) induced electric field-based biochip platform, which has an open-top sub-mm square chamber enclosed by four sidewall electrodes and two bottom electrodes, to achieve rotation about the two axes, thus 3D cell rotation. By applying an ac potential to the four sidewall electrodes, an in-plane (yaw) rotating electric field is generated and in-plane rotation is achieved. Similarly, by applying an ac potential to two opposite sidewall electrodes and the two bottom electrodes, an out-of-plane (pitch) rotating electric field is generated and rolling rotation is achieved. As a prompt proof-of-concept, bottom electrodes were constructed with transparent indium tin oxide (ITO) using the standard lift-off process and the sidewall electrodes were constructed using a low-cost micro-milling process and then assembled to form the chip. Through experiments, we demonstrate rotation of bovine oocytes of ~120 µm diameter about two axes, with the capability of controlling the rotation direction and the rate for each axis through control of the ac potential amplitude, frequency, and phase shift, and cell medium conductivity. The maximum observed rotation rate reached nearly 140° s⁻¹, while a consistent rotation rate reached up to 40° s⁻¹. Rotation rate spectra for zona pellucida-intact and zona pellucida-free oocytes were further compared and found to have no effective difference. This simple, transparent, cheap-to-manufacture, and open-top platform allows additional functional modules to be integrated to become a more powerful cell manipulation system.

Electrical bioimpedance measurement as a tool for dysphagia visualisation.

A non-invasive and portable bioimpedance method and a device for detecting superior to inferior closure of the pharynx during swallowing have been developed. The 2-channel device measures electric impedance across the neck at two levels of the pharynx via injected currents at 40 and 70 kHz. The device has been trialled on both healthy and dysphagic subjects. Results from these trials revealed a relationship (r = 0.59) between the temporal separation of the second peaks in the bioimpedance waveforms and descending pressure sequence in the pharynx as measured by pharyngeal manometry. However, these features were only clearly visible in the bioimpedance waveforms for 64% of swallows. Further research is underway to improve the bioimpedance measurement reliability and validate waveform feature correlation to swallowing to maximise the device's efficacy in dysphagia rehabilitation.