Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report that the relative intensities in each plane of excitation depend on the Stokes shift of the fluorochrome. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ~90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.Comment: 15 pages, 4 figures; changed the discussion of narrow-band detected fringes (Fig. 3) to describe the phenomenon as a moire pattern between the excitation and emission standing-wave fields, rather than a beats pattern; added DiI(5)-labelled red blood cell in Fig. 4 to show that standing-wave fringes are present even when the dye excitation wavelength is outside the haemoglobin absorption ban

Device selection algorithm for mobile traffic offloading

Aim of thesis is to reduce the data traffic from mobile network by exploiting Ad-hoc modes of communication. This can be achieved by sending message to few mobile nodes which will forward data to rest of the mobile nodes via Ad-hoc communication. This thesis focuses on selection of those few mobile nodes (Target Set) which will receive message directly from mobile network and can effectively reduce the data traffic on mobile network by forwarding that message to many other mobile nodes via Ad-hoc communication. Thesis consists of two tasks. First task of thesis is to implement the concept of Mobile Traffic Offloading on android based smart phones using desktop as server and two smart phones as its clients. Second task of the thesis is to implement the same concept on a large scale with more scope for Target Set selection. Second task consists of five different approaches for the selection of Target Set of mobile. First approach is Random approach, in this approach server sends message to a randomly selected Target Set of mobile nodes from all available mobile nodes. Second and third approaches are Distance and Distance Extended. These approaches considers the euclidean distance between each pair of mobile nodes. Distance approach considers the current euclidean distance and checks the possibility of Ad-hoc communication. Distance Extended approach uses the current distance between the mobile nodes and also calculates the probabilistic future distance between mobile nodes. Fourth approach is Same Street, this approach considers the position of mobile nodes on the street map and uses that position to find Target Set of mobile nodes. Final approach of thesis is Path Length approach. This approach also is a map based approach and finds shortest distance between each pair of mobile nodes. This approach also considers the probability of Ad-hoc communication in future. Finally this thesis presents the evaluation for both tasks. Evaluation of first task shows that how message delay changes with increase in message size. Evaluation of second task compares all Target Set selection approaches on the basis of number of Ad-hoc messages, Message Delay and Energy Consumption .Thesis also compares the performance of advance approaches(Distance Extended and Path Length) on increasing the Ad-hoc range of communication

Optical fibre-tip probes for SERS: numerical study for design considerations.

Enhancement of sub-wavelength optical fields using sub-micron plasmonic probes has found many applications in chemical, material, biological and medical sciences. The enhancement is via localised surface-plasmon resonance (LSPR) which enables the highly sensitive vibrational-spectroscopy technique of surface-enhanced Raman scattering (SERS). Combining SERS with optical fibres can allow the monitoring of biochemical reactions in situ with high resolution. Here, we study the electromagnetic-field enhancement of a tapered optical fibre-tip coated with gold nanoparticles (AuNPs) using finite-element simulations. We investigate the electric-field enhancement associated with metallic NPs and study the effect of parameters such as tip-aperture radius, cone angle, nanoparticle size and gaps between them. Our study provides an understanding of the design and application of metal-nanoparticle-coated optical-fibre-tip probes for SERS. The approach of using fibre-coupled delivery adds flexibility and simplifies the system requirements in SERS, making it suitable for cellular imaging and mapping bio-interfaces.Trinity College, Cambridge for a PhD studentship Darwin College, Cambridge for a Henslow Research Fellowshi

Optimising superoscillatory spots for far-field super-resolution imaging

Optical superoscillatory imaging, allowing unlabelled far-field super-resolution, has in recent years become reality. Instruments have been built and their super-resolution imaging capabilities demonstrated. The question is no longer whether this can be done, but how well: what resolution is practically achievable? Numerous works have optimised various particular features of superoscillatory spots, but in order to probe the limits of superoscillatory imaging we need to simultaneously optimise all the important spot features: those that define the resolution of the system. We simultaneously optimise spot size and its intensity relative to the sidebands for various fields of view, giving a set of best compromises for use in different imaging scenarios. Our technique uses the circular prolate spheroidal wave functions as a basis set on the field of view, and the optimal combination of these, representing the optimal spot, is found using a multi-objective genetic algorithm. We then introduce a less computationally demanding approach suitable for real-time use in the laboratory which, crucially, allows independent control of spot size and field of view. Imaging simulations demonstrate the resolution achievable with these spots. We show a three-order-of-magnitude improvement in the efficiency of focusing to achieve the same resolution as previously reported results, or a 26 % increase in resolution for the same efficiency of focusing

Investigating biomechanical noise in neuroblastoma cells using the quartz crystal microbalance.

Quantifying cellular behaviour by motility and morphology changes is increasingly important in formulating an understanding of fundamental physiological phenomena and cellular mechanisms of disease. However, cells are complex biological units, which often respond to external environmental factors by manifesting subtle responses that may be difficult to interpret using conventional biophysical measurements. This paper describes the adaptation of the quartz crystal microbalance (QCM) to monitor neuroblastoma cells undergoing environmental stress wherein the frequency stability of the device can be correlated to changes in cellular state. By employing time domain analysis of the resulting frequency fluctuations, it is possible to study the variations in cellular motility and distinguish between different cell states induced by applied external heat stress. The changes in the frequency fluctuation data are correlated to phenotypical physical response recorded using optical microscopy under identical conditions of environmental stress. This technique, by probing the associated biomechanical noise, paves the way for its use in monitoring cell activity, and intrinsic motility and morphology changes, as well as the modulation resulting from the action of drugs, toxins and environmental stress.This is the author accepted manuscript. The final version is available from Royal Society Publishing via http://dx.doi.org/10.1098/rsif.2014.138

Nanoparticles and intracellular applications of surface-enhanced Raman spectroscopy

Surface-enhanced Raman spectroscopy (SERS) offers ultrasensitive vibrational fingerprinting at the nanoscale. Its non-destructive nature affords an ideal tool for interrogation of the intracellular environment, detecting the localisation of biomolecules, delivery and monitoring of therapeutics and for characterisation of complex cellular processes at the molecular level. Innovations in nanotechnology have produced a wide selection of novel, purpose-built plasmonic nanostructures capable of high SERS enhancement for intracellular probing while microfluidic technologies are being utilised to reproducibly synthesise nanoparticle (NP) probes at large scale and in high throughput. Sophisticated multivariate analysis techniques unlock the wealth of previously unattainable biomolecular information contained within large and multidimensional SERS datasets. Thus, with suitable combination of experimental techniques and analytics, SERS boasts enormous potential for cell based assays and to expand our understanding of the intracellular environment. In this review we trace the pathway to utilisation of nanomaterials for intracellular SERS. Thus we review and assess nanoparticle synthesis methods, their toxicity and cell interactions before presenting significant developments in intracellular SERS methodologies and how identified challenges can be addressed

Visualizing electromagnetic fields at the nanoscale by single molecule localization.

Coupling of light to the free electrons at metallic surfaces allows the confinement of electric fields to subwavelength dimensions, far below the optical diffraction limit. While this is routinely used to manipulate light at the nanoscale, in electro-optic devices and enhanced spectroscopic techniques, no characterization technique for imaging the underlying nanoscopic electromagnetic fields exists, which does not perturb the field or employ complex electron beam imaging. Here, we demonstrate the direct visualization of electromagnetic fields on patterned metallic substrates at nanometer resolution, exploiting a strong "autonomous" fluorescence-blinking behavior of single molecules within the confined fields allowing their localization. Use of DNA-constructs for precise positioning of fluorescence dyes on the surface induces this distance-dependent autonomous blinking thus completely obviating the need for exogenous agents or switching methods. Mapping such electromagnetic field distributions at nanometer resolution aids the rational design of nanometals for diverse photonic applications.We acknowledge financial support from EPSRC grant EP/G060649/1, EP/H028757/1-2, EP/I012060/1, EP/L015889/1, MRC grant MR/K015850/1 and ERC grant LINASS 320503.This is the author accepted manuscript. The final version is available from ACS at http://pubs.acs.org/doi/abs/10.1021/acs.nanolett.5b00405

Identification of microplastics in a large water volume by integrated holography and Raman spectroscopy

Funding Japan Science and Technology Agency SICORP (JPMJSC1705); Natural Environment Research Council (NE/R01227X/1); Kajima Foundation (Overseas research grant); Japan Society for the Promotion of Science (18H03810, 18K13934); Kurita Water and Environment Foundation (17B030).Peer reviewedPublisher PD

Multimodal Image and Spectral Feature Learning for Efficient Analysis of Water-Suspended Particles

apan Science and Technology Agency SICORP and Natural Environment Research Council (JST-NERC SICORP Marine Sensor Proof of Concept Grant JPMJSC1705, NE/R01227X/1); JSPS KAKENHI Grant (18K13934 and 18H03810); Sumitomo Foundation: Grant for environmental Research Project (203122). Acknowledgments. The authors thank Dr. T. Fukuba for the support for building the experimental setup. The authors also thank Dr. H. Sawada for providing samples for this work.Peer reviewedPublisher PD

Observing Single Molecules Complexing with Cucurbit[7]uril through Nanogap Surface-Enhanced Raman Spectroscopy.

In recent years, single-molecule sensitivity achievable by surface-enhanced Raman spectroscopy (SERS) has been widely reported. We use this to investigate supramolecular host-guest chemistry with the macrocyclic host cucurbit[7]uril, on a few-to-single-molecule level. A nanogap geometry, comprising individual gold nanoparticles on a planar gold surface spaced by a single layer of molecules, gives intense SERS signals. Plasmonic coupling between the particle and the surface leads to strongly enhanced optical fields in the gap between them, with single-molecule sensitivity established using a modification of the well-known bianalyte method. Changes in the Raman modes of the host molecule are observed when single guests included inside its cavity internally stretch it. Anisotropic intermolecular interactions with the guest are found which show additional distinct features in the Raman modes of the host molecule.The authors acknowledge funding from Walters-Kundert Trust, EPSRC (EP/K028510/1, EP/G060649/1, EP/ H007024/1, ERC LINASS 320503), an ERC starting investigator grant (ASPiRe 240629), EU CUBiHOLE grant and the Defence Science and Technology Laboratory (DSTL). S.K. thanks Krebs Memorial Scholarship (The Biochemical Society) and Cambridge Commonwealth Trust for funding.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acs.jpclett.5b0253