Dynamical hologram generation for high speed optical trapping of smart droplet microtools

This paper demonstrates spatially selective sampling of the plasma membrane by the implementation of time-multiplexed holographic optical tweezers for Smart Droplet Microtools (SDMs). High speed (>1000fps) dynamical hologram generation was computed on the graphics processing unit of a standard display card and controlled by a user friendly LabView interface. Time multiplexed binary holograms were displayed in real time and mirrored to a ferroelectric Spatial Light Modulator. SDMs were manufactured with both liquid cores (as previously described) and solid cores, which confer significant advantages in terms of stability, polydispersity and ease of use. These were coated with a number of detergents, the most successful based upon lipids doped with transfection reagents. In order to validate these, trapped SDMs were maneuvered up to the plasma membrane of giant vesicles containing Nile Red and human biliary epithelial (BE) colon cancer cells with green fluorescent labeled protein (GFP)-labeled CAAX (a motif belonging to the Ras protein). Bright field and fluorescence images showed that successful trapping and manipulation of multiple SDMs in x, y, z was achieved with success rates of 30-50% and that subsequent membrane-SDM interactions led to the uptake of Nile Red or GFP-CAAX into the SDM

A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs)

We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP

Multidimensional luminescence microscope for imaging defect colour centres in diamond

We report a multidimensional luminescence microscope providing hyperspectral imaging and time-resolved (luminescence lifetime) imaging for the study of luminescent diamond defects. The instrument includes crossed-polariser white light transmission microscopy to reveal any birefringence that would indicate strain in the diamond lattice. We demonstrate the application of this new instrument to defects in natural and synthetic diamonds including N3, nitrogen and silicon vacancies. Hyperspectral imaging provides contrast that is not apparent in conventional intensity images and the luminescence lifetime provides further contrast

Multidimensional spectroscopy and imaging of defects in synthetic diamond: excitation-emission-lifetime luminescence measurements with multiexponential fitting and phasor analysis

We report the application of phasor analysis and nonlinear iterative fitting to complex spatial and spectroscopic luminescence decay data obtained from multidimensional microscopy of a CVD diamond grown on a HPHT substrate. This spectral and lifetime-resolved analysis enabled spatial mapping of variations in concentrations of nitrogen vacancy (NV) defects in both charge states and the quenching of NV− defects, as well as the identification of SiV− luminescence. These imaging and spectroscopic modalities may be important for reliable fabrication of quantum devices based on such defects in diamond, which will require well-defined and characterised quantum electronic properties

An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy

We demonstrate that spectral selection from a supercontinuum generated in a microstructured fibre can provide a continuously electronically tunable ultrafast spatially coherent source for confocal microscopy and both scanning and wide field fluorescence lifetime imaging. © 2005 Optical Society of America

An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy

Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off ' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems