Label-free Brillouin endo-microscopy for the quantitative 3D imaging of sub-micrometre biology.

This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 µm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level.

Impedimetric Characterization of Bipolar Nanoelectrodes with Cancer Cells.

Merging of electronics with biology, defined as bioelectronics, at the nanoscale holds considerable promise for sensing and modulating cellular behavior. Advancing our understanding of nanobioelectronics will facilitate development and enable applications in biosensing, tissue engineering, and bioelectronic medicine. However, studies investigating the electrical effects when merging wireless conductive nanoelectrodes with biology are lacking. Consequently, a tool is required to develop a greater understanding of merging conductive nanoparticles with cells. Herein, this challenge is addressed by developing an impedimetric method to evaluate bipolar electrode (BPE) systems that could report on electrical input. A theoretical framework is provided, using impedance to determine if conductive nanoparticles can be polarized and used to drive current. It is then demonstrated that 125 nm of gold nanoparticle (AuNP) bipolar electrodes (BPEs) could be sensed in the presence of cells when incorporated intracellularly at 500 µg/mL using water and phosphate-buffered saline (PBS) as electrolytes. These results highlight how nanoscale BPEs act within biological systems. This research will impact the rational design of using BPE systems in cells for both sensing and actuating applications.

Plasmonic-based impedance microspectroscopy of optically heterogeneous samples.

A robust impedance microscopy technique is presented. This optical tool enables high resolution imaging of electrical properties with promising biophysical applications. The underlying principle is that surface plasmon resonance (SPR) sensors are able to measure perturbations of surface charge density and therefore can be used to compute the impedance of surface-adhered cells. However, the ability to perform reliable quantitative impedance imaging is affected by the optical heterogeneity of the cell-sensor interface. To address this issue, a novel method for quantitative time-resolved resonance angle tracking is developed and applied to correct for the effect of the optical properties. To demonstrate the capability of this technique, impedance microspectroscopy of bovine serum albumin (BSA) patterns was performed enabling measurements of capacitance with submicroscopic resolution. The work presented offers an impedance microspectroscopy method that will create new avenues in studying the electrical properties of single cells and biomolecules as well as bio-electrical currents.

Surface plasmon resonance imaging of excitable cells.

Surface plasmons (SPs) are surface charge density oscillations occuring at a metal/dieletric interface and are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. SPR imaging systems using high numerical aperture (NA) objective lenses have been designed to image adherent live cells with high magnification and near-diffraction limited spatial resolution. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained. In this paper, we present the development of a multimodal microscope that combines SPR imaging with a number of additional imaging modalities including bright-field, epifluorescence, total internal reflection microscopy and SPR fluorescence microscopy. We used a high NA objective lens for SPR and TIR microscopy and the platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. Both the SPR and TIR imaging systems feature a wide field of view (~300 µm) that allows measurements from multiple cells whilst maintaining a resolution sufficient to image fine cellular processes. The capability of the platform to perform label-free functional imaging of living cells was demonstrated by imaging the spatial variations in contractions from stem cell-derived cardiomyocytes. This technique shows promise for non-invasive imaging of cultured cells over very long periods of time during development.

Rapid one-step biotinylation of biological and non-biological surfaces.

We describe a rapid one-step method to biotinylate virtually any biological or non-biological surface. Contacting a solution of biotin-spacer-lipid constructs with a surface will form a coating within seconds on non-biological surfaces or within minutes on most biological membranes including membrane viruses. The resultant biotinylated surface can then be used to interact with avidinylated conjugates, beads, vesicles, surfaces or cells.

Sensitive detection of voltage transients using differential intensity surface plasmon resonance system.

This paper describes theoretical and experimental study of the fundamentals of using surface plasmon resonance (SPR) for label-free detection of voltage. Plasmonic voltage sensing relies on the capacitive properties of metal-electrolyte interface that are governed by electrostatic interactions between charge carriers in both phases. Externally-applied voltage leads to changes in the free electron density in the surface of the metal, shifting the SPR position. The study shows the effects of the applied voltage on the shape of the SPR curve. It also provides a comparison between the theoretical and experimental response to the applied voltage. The response is presented in a universal term that can be used to assess the voltage sensitivity of different SPR instruments. Finally, it demonstrates the capacity of the SPR system in resolving dynamic voltage signals; a detection limit of 10mV with a temporal resolution of 5ms is achievable. These findings pave the way for the use of SPR systems in the detection of electrical activity of biological cells.