Fully angularly resolved 3D microrheology with optical tweezers.

Microrheology with optical tweezers (MOT) is an all-optical technique that allows the user to investigate a materials' viscoelastic properties at microscopic scales, and is particularly useful for those materials that feature complex microstructures, such as biological samples. MOT is increasingly being employed alongside 3D imaging systems and particle tracking methods to generate maps showing not only how properties may vary between different points in a sample but also how at a single point the viscoelastic properties may vary with direction. However, due to the diffraction limited shape of focussed beams, optical traps are inherently anisotropic in 3D. This can result in a significant overestimation of the fluids' viscosity in certain directions. As such, the rheological properties can only be accurately probed along directions parallel or perpendicular to the axis of trap beam propagation. In this work, a new analytical method is demonstrated to overcome this potential artefact. This is achieved by performing principal component analysis on 3D MOT data to characterise the trap, and then identify the frequency range over which trap anisotropy influences the data. This approach is initially applied to simulated data for a Newtonian fluid where the trap anisotropy induced maximum error in viscosity is reduced from ~ 150% to less than 6%. The effectiveness of the method is corroborated by experimental MOT measurements performed with water and gelatine solutions, thus confirming that the microrheology of a fluid can be extracted reliably across a wide frequency range and in any arbitrary direction. This work opens the door to fully spatially and angularly resolved 3D mapping of the rheological properties of soft materials over a broad frequency range.

Metasurface for Engineering Superimposed Ince-Gaussian Beams.

Ince-Gaussian beams (IGBs) are the third complete family of exact and orthogonal solutions of the paraxial wave equation and have been applied in many fields ranging from particle trapping to quantum optics. IGBs play a very important role in optics as they represent the exact and continuous transition modes connecting Laguerre-Gaussian and Hermite-Gaussian beams. The method currently in use suffers from the high cost, complexity, and large volume of the optical system. The superposition of IGBs can generate complicated structured beams with multiple phase and polarization singularities. A metasurface approach is proposed to realizing various superpositions of IGBs without relying on a complicated optical setup. By superimposing IGBs with even and odd modes, multiple phase, and polarization singularities are observed in the resultant beams. The phase and polarization singularities are modulated by setting the initial phase in the design and controlling the incident linear polarization. The compactness of the developed metasurface devices and the unique properties of the generated beams have the potential to impact many practical applications such as particle manipulation, orbital angular momentum spectrum manipulation, and optical communications.

OptoRheo: Simultaneous in situ micro-mechanical sensing and imaging of live 3D biological systems.

Biomechanical cues from the extracellular matrix (ECM) are essential for directing many cellular processes, from normal development and repair, to disease progression. To better understand cell-matrix interactions, we have developed a new instrument named 'OptoRheo' that combines light sheet fluorescence microscopy with particle tracking microrheology. OptoRheo lets us image cells in 3D as they proliferate over several days while simultaneously sensing the mechanical properties of the surrounding extracellular and pericellular matrix at a sub-cellular length scale. OptoRheo can be used in two operational modalities (with and without an optical trap) to extend the dynamic range of microrheology measurements. We corroborated this by characterising the ECM surrounding live breast cancer cells in two distinct culture systems, cell clusters in 3D hydrogels and spheroids in suspension culture. This cutting-edge instrument will transform the exploration of drug transport through complex cell culture matrices and optimise the design of the next-generation of disease models.

Trophic Transfer of Single-Walled Carbon Nanotubes at the Base of the Food Chain and Toxicological Response.

The potential for trophic transfer of single-walled carbon nanotubes (SWCNTs) was assessed using the green algae Tetraselmis suecica and the blue mussel Mytilus edulis in a series of laboratory experiments. Swanee River Natural Organic Matter (SRNOM)-dispersed SWCNTs were introduced into growing algal cultures. Light microscopical observations, confirmed by scanning electronic microscopy (SEM) and Raman spectroscopy, showed that SWCNT agglomerates adhered to the external algal cell walls and transmission electronic microscopy (TEM) results suggested internalization. A direct effect of SWCNT exposure on the algae was a significant decrease in growth, expressed as chlorophyll a concentration and cell viability. Mussels, fed with algae in the presence of SWCNTs, led to significantly increased pseudofaeces production, indicating selective feeding. Nevertheless, histological sections of the mussel digestive gland following exposure showed evidence of SWCNT-containing algae. Furthermore, DNA damage and oxidative stress biomarker responses in the mussel haemocytes and gill tissue were significantly altered from baseline values and were consistent with previously observed responses to SWCNT exposure. In conclusion, the observed SWCNT-algal interaction demonstrated the potential for SWCNT entrance at the base of the food chain, which may facilitate their trophic transfer with potential consequences for human exposure and health.

Optical Tweezers with Integrated Multiplane Microscopy (OpTIMuM): a new tool for 3D microrheology.

We introduce a novel 3D microrheology system that combines for the first time Optical Tweezers with Integrated Multiplane Microscopy (OpTIMuM). The system allows the 3D tracking of an optically trapped bead, with ~ 20 nm accuracy along the optical axis. This is achieved without the need for a high precision z-stage, separate calibration sample, nor a priori knowledge of either the bead size or the optical properties of the suspending medium. Instead, we have developed a simple yet effective in situ spatial calibration method using image sharpness and exploiting the fact we image at multiple planes simultaneously. These features make OpTIMuM an ideal system for microrheology measurements, and we corroborate the effectiveness of this novel microrheology tool by measuring the viscosity of water in three dimensions, simultaneously.

Fabrication and characterization of machined multi-core fiber tweezers for single cell manipulation: erratum.

We have found an error in our work reported in [Opt. Express26(3), 3557 (2018)], which we correct in this erratum. We used incorrect data for the experimentally measured values of power of the fibre trap and power of the conventional optical tweezers (OT) used to 'break' the fibre trap. Using the correct data, Ffibre and Q (force and quality) of the multicore fibre tweezer are re-calculated. In this erratum, we communicate the correct values of Ffibre and Q and publish a revised Fig. 7 that contains results based on the correct data. Based on the revised result, two statements, in the abstract and conclusions, are also revised. The fabrication method, technique and general conclusion remain unaffected.

Publisher Correction: Femtosecond laser fabrication of silver nanostructures on glass for surface enhanced Raman spectroscopy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Femtosecond laser fabrication of silver nanostructures on glass for surface enhanced Raman spectroscopy.

We report on an optimized fabrication protocol for obtaining silver nanoparticles on fused silica substrates via laser photoreduction of a silver salt solution. We find that multiple scans of the laser over the surface leads to a more uniform coverage of densely packed silver nanoparticles of approximately 50 nm diameter on the fused silica surface. Our substrates yield Raman enhancement factors of the order of 1011 of the signal detected from crystal violet. We use a theoretical model based on scanning electron microscope (SEM) images of our substrates to explain our experimental results. We also demonstrate how our technique can be extended to embedding silver nanoparticles in buried microfluidic channels in glass. The in situ laser inscription of silver nanoparticles on a laser machined, sub-surface, microfluidic channel wall within bulk glass paves the way for developing 3D, monolithic, fused silica surface enhance Raman spectroscopy (SERS) microfluidic sensing devices.

Single Cell Isolation Using Optical Tweezers.

Optical tweezers offer a non-contact method for selecting single cells and translocating them from one microenvironment to another. We have characterized the optical tweezing of yeast S. cerevisiae and can manipulate single cells at 0.41 ± 0.06 mm/s using a 26.8 ± 0.1 mW from a 785 nm diode laser. We have fabricated and tested three cell isolation devices; a micropipette, a PDMS chip and a laser machined fused silica chip and we have isolated yeast, single bacteria and cyanobacteria cells. The most effective isolation was achieved in PDMS chips, where single yeast cells were grown and observed for 18 h without contamination. The duration of budding in S. cerevisiae was not affected by the laser parameters used, but the time from tweezing until the first budding event began increased with increasing laser energy (laser power × time). Yeast cells tweezed using 25.0 ± 0.1 mW for 1 min were viable after isolation. We have constructed a micro-consortium of yeast cells, and a co-culture of yeast and bacteria, using optical tweezers in combination with the PDMS network of channels and isolation chambers, which may impact on both industrial biotechnology and understanding pathogen dynamics.

Fabrication and characterization of machined multi-core fiber tweezers for single cell manipulation.

Optical tweezing is a non-invasive technique that can enable a variety of single cell experiments; however, it tends to be based on a high numerical aperture (NA) microscope objective to both deliver the tweezing laser light and image the sample. This introduces restrictions in system flexibility when both trapping and imaging. Here, we demonstrate a novel, high NA tweezing system based on micro-machined multicore optical fibers. Using the machined, multicore fiber tweezer, cells are optically manipulated under a variety of microscopes, without requiring a high NA objective lens. The maximum NA of the fiber-based tweezer demonstrated is 1.039. A stable trap with a maximum total power 30 mW has been characterized to exert a maximum optical force of 26.4 pN, on a trapped, 7 µm diameter yeast cell. Single cells are held 15-35 µm from the fiber end and can be manipulated in the x, y and z directions throughout the sample. In this way, single cells are controllably trapped under a Raman microscope to categorize the yeast cells as live or dead, demonstrating trapping by the machined multicore fiber-based tweezer decoupled from the imaging or excitation objective lens.

Natural marine bacteria as model organisms for the hazard-assessment of consumer products containing silver nanoparticles.

Scarce information is available regarding the fate and toxicology of engineered silver nanoparticles (AgNPs) in the marine environment, especially when compared to other environmental compartments. Hence, the antibacterial activity of the NM-300 AgNPs (OECD programme) and a household product containing colloidal AgNPs (Mesosilver) was investigated using marine bacteria, pure cultures and natural mixed populations (microcosm approach). Bacterial susceptibility to AgNPs was species-specific, with Gram negative bacteria being more resistant than the Gram positive species (NM-300 concentration used ranged between 0.062 and 1.5 mg L-1), and the Mesosilver product was more toxic than the NM-300. Bacterial viability and the physiological status (O2 uptake measured by respirometry) of the microbial community in the microcosm was negatively affected at an initial concentration of 1 mg L-1 NM-300. The high chloride concentrations in the media/seawater led to the formation of silver-chloro complexes thus enhancing AgNP toxicity. We recommend the use of natural marine bacteria as models when assessing the environmental relevant antibacterial properties of products containing nanosilver.

Determining the 3D orientation of optically trapped upconverting nanorods by in situ single-particle polarized spectroscopy.

An approach to unequivocally determine the three-dimensional orientation of optically manipulated NaYF4:Er(3+),Yb(3+) upconverting nanorods (UCNRs) is demonstrated. Long-term immobilization of individual UCNRs inside single and multiple resonant optical traps allow for stable single UCNR spectroscopy studies. Based on the strong polarization dependent upconverted luminescence of UCNRs it is possible to unequivocally determine, in real time, their three-dimensional orientation when optically trapped. In single-beam traps, polarized single particle spectroscopy has concluded that UCNRs orientate parallel to the propagation axis of the trapping beam. On the other hand, when multiple-beam optical tweezers are used, single particle polarization spectroscopy demonstrated how full spatial control over UCNR orientation can be achieved by changing the trap-to-trap distance as well as the relative orientation between optical traps. All these results show the possibility of real time three-dimensional manipulation and tracking of anisotropic nanoparticles with wide potential application in modern nanobiophotonics.

Shifts in the metabolic function of a benthic estuarine microbial community following a single pulse exposure to silver nanoparticles.

The increasing use of silver nanoparticles (AgNPs) as a biocidal agent and their potential accumulation in sediments may threaten non-target natural environmental bacterial communities. In this study a microcosm approach was established to investigate the effects of well characterized OECD AgNPs (NM-300) on the function of the bacterial community inhabiting marine estuarine sediments (salinity 31‰). The results showed that a single pulse of NM-300 AgNPs (1 mg L(-1)) that led to sediment concentrations below 6 mg Ag kg(-1) dry weight inhibited the bacterial utilization of environmentally relevant carbon substrates. As a result, the functional diversity changed, but recovered after 120 h under the experimental conditions. This microcosm study suggests that AgNPs under environmentally relevant experimental conditions can negatively affect bacterial function and provides an insight into the understanding of the bacterial community response and resilience to AgNPs exposure, important for informing relevant regulatory measures.

Potentiating toxicological interaction of single-walled carbon nanotubes with dissolved metals.

The present study explored the ecotoxicology of single-walled carbon nanotubes (SWCNTs) and their likely interaction with dissolved metals, with a focus on the effect of in vivo exposure in marine mussels. Any nano-scale effects were negated by the tendency of uncoated SWCNTs to agglomerate in water, particularly with high ionic strength as is the case in estuarine and full-strength seawater. However, SWCNTs, in combination with natural organic matter, remained suspended in seawater for long enough to become available to filter-feeding mussels, leading to their concentration on and increased contact with gill epithelia during exposure. For the first time, the authors describe a potentiating toxicological effect, expressed as DNA strand breaks obtained using the comet assay, on divalent metals afforded by negatively charged SWCNT agglomerates in seawater at concentrations as low as 5 µg L⁻¹. This is supported by the observation that SWCNTs alone were only toxic at concentrations ≥100 µg L⁻¹ and that the SWCNT-induced DNA damage was correlated with oxidative stress only in the absence of metals. If these laboratory experiments are confirmed in the natural environment, the present results will have implications for the understanding of the role of carbon nanotubes in environmental metal dynamics, toxicology, and consequently, regulatory requirements.

Quantum dot-based thermal spectroscopy and imaging of optically trapped microspheres and single cells.

Laser-induced thermal effects in optically trapped microspheres and single cells are investigated by quantum dot luminescence thermometry. Thermal spectroscopy has revealed a non-localized temperature distribution around the trap that extends over tens of micrometers, in agreement with previous theoretical models besides identifying water absorption as the most important heating source. The experimental results of thermal loading at a variety of wavelengths reveal that an optimum trapping wavelength exists for biological applications close to 820 nm. This is corroborated by a simultaneous analysis of the spectral dependence of cellular heating and damage in human lymphocytes during optical trapping. This quantum dot luminescence thermometry demonstrates that optical trapping with 820 nm laser radiation produces minimum intracellular heating, well below the cytotoxic level (43 °C), thus, avoiding cell damage.

Quantum dot enabled thermal imaging of optofluidic devices.

Quantum dot thermal imaging has been used to analyse the chromatic dependence of laser-induced thermal effects inside optofluidic devices with monolithically integrated near-infrared waveguides. We demonstrate how microchannel optical local heating plays an important role, which cannot be disregarded within the context of on-chip optical cell manipulation. We also report on the thermal imaging of locally illuminated microchannels when filled with nano-heating particles such as carbon nanotubes.

A 3D mammalian cell separator biochip.

The dissimilar cytoskeletal architecture in diverse cell types induces a difference in their deformability that presents a viable approach to separate cells in a non-invasive manner. We report on the design and fabrication of a robust and scalable device capable of separating a heterogeneous population of cells with variable degree of deformability into enriched populations with deformability above a certain threshold. The three dimensional device was fabricated in fused silica by femtosecond laser direct writing combined with selective chemical etching. The separator device was evaluated using promyelocytic HL60 cells. Using flow rates as large as 167 µL min(-1), throughputs of up to 2800 cells min(-1) were achieved at the device output. A fluorescence-activated cell sorting (FACS) viability analysis on the cells revealed 81% of the population maintain cellular integrity after passage through the device.

Multiplane imaging and three dimensional nanoscale particle tracking in biological microscopy.

A conventional microscope produces a sharp image from just a single object-plane. This is often a limitation, notably in cell biology. We present a microscope attachment which records sharp images from several object-planes simultaneously. The key concept is to introduce a distorted diffraction grating into the optical system, establishing an order-dependent focussing power in order to generate several images, each arising from a different object-plane. We exploit this multiplane imaging not just for bio-imaging but also for nano-particle tracking, achieving approximately 10 nm z position resolution by parameterising the images with an image sharpness metric.

Passive optical separation within a 'nondiffracting' light beam.

A passive, optical cell sorter is created using the light pattern of a 'nondiffracting' beam-the Bessel beam. As a precursor to cell sorting studies, microspheres are used to test the resolution of the sorter on the basis of particle size and refractive index. Variations in size and, more noticeably, refractive index, lead to a marked difference in the migration time of spheres in the Bessel beam. Intrinsic differences (size, refractive index) between native (unlabeled) cell populations are utilized for cell sorting. The large difference in size between erythrocytes and lymphocytes results in their successful separation in this beam pattern. The intrinsic differences in size and refractive index of other cells in the study (HL60 human promyelocytic leukaemic cells, murine bone marrow, and murine stem/progenitor cells) are not large enough to induce passive optical separation. Silica microsphere tags are attached to cells of interest to modify their size and refractive index, resulting in the separation of labeled cells. Cells collected after separation are viable, as evidenced by trypan blue dye exclusion, their ability to clone in vitro, continued growth in culture, and lack of expression of Caspase 3, a marker of apoptosis.