Andrew D. Ward

Raman tweezers sorting of single microbial cells

We have selectively isolated microbial cells by identifying and then manipulating cells using a combination of Raman microspectroscopy and optical trapping. The criterion for cell discrimination is based on spectral peak shifts within the Raman spectrum of individual cells. A specific shift in the phenylalanine peak position from 1001 rel. cm(-1) to 965 rel. cm(-1) is utilized to indicate the uptake of (13) C within the cell that utilized (13) C-substrate. Cells were captured and manipulated using an infrared (1064 nm) laser while Raman spectra were acquired over shorter timescales (30 s) using a co-aligned 514.5 nm laser beam. Selected cells were manoeuvred to a clean part of a capillary tube and the tubes were cleaved to physically separate the cells. The technique was tested for cell viability and cross-contamination effects using 70 single yeast cells (Saccharomyces cerevisia). Following these tests, 58 single bacterial cells (Escherichia coli DH5α, and Pseudomonas fluorescens SBW25::Km-RFP) that exhibited (13) C uptake were sorted from bacterial populations. Among those isolated cells, 11 out of 18 yeast cells and 7 out of 18 single SBW25::Km-RFP cells were recovered by incubation; 2 out of 7 sorted yeast cells and 3 out of 8 sorted bacterial cells (single SBW25::Km-RFP) were genome amplified correctly. We show that the Raman tweezers approach has the potential to open a new frontier to study unculturable microorganisms, which account for more than 99% microbes in natural environment.

Spectral discrimination of live prostate and bladder cancer cell lines using Raman optical tweezers

An investigation into the use of Raman optical tweezers to study urological cell lines is reported, with the ultimate aim of determining the presence of malignant CaP cells in urine and peripheral fluids. To this end, we trapped and analyzed live CaP cells (PC-3) and bladder cells (MGH-U1), because both prostate and bladder cells are likely to be present in urine. The laser excitation wavelength of 514.5 nm was used, with Raman light collected both in back- and forward-scattering geometric configurations. For the backscattering configuration the same laser was used for trapping and excitation, while for forward scattering a 1064 nm laser provided the trapping beam. Analysis of cell-diameter distributions for cells analyzed suggested normal distribution of cell sizes, indicating an unbiased cell-selection criterion. Principal components analysis afforded discrimination of MGH-U1 and PC-3 spectra collected in either configuration, demonstrating that it is possible to trap, analyze, and differentiate PC-3 from MGH-U1 cells using a 514.5 nm laser. By loading plot analysis, possible biomolecules responsible for discrimination in both configurations were determined. Finally, the effect of cell size on discrimination was investigated, with results indicating that separation is based predominantly on cell type rather than cell size.

Classification of fixed urological cells using Raman tweezers

In this paper we report on preliminary investigations into using Raman tweezers to classify urological cell lines. This builds on earlier work within the group, whereby Raman tweezer methodologies were developed, and the application of this technique to differentiate between live prostate cancer (CaP) and bladder cells lines (PC-3 and MGH-U1 respectively) was demonstrated.In this present study we analysed chemically fixed cells using two different fixative methods; SurePath (a commercial available liquid based cytology media) and 4% v/v formalin/PBS fixatives. The study has been expanded from our previous live cell study to include the androgen sensitive CaP cell line LNCaP, primary benign prostate hyperplasia (BPH) cells as well as primary urethral cells. Raman light from the cells was collected using a 514.5 nm Ar-ion laser excitation source in back-scattering configuration mode.Principal component-linear discriminate analysis (PC-LDA) models of resulting cell spectra were generated and these were validated using a blind comparison. Sensitivities and specificities of > 72% and 90% respectively, for SurePath fixed cells, and > 93% and 98% respectively for 4% v/v formalin/PBS fixed cells was achieved. The higher prediction results for the formalin fixed cells can be attributed to a better signal-to-noise ratio for spectra obtained from these cells.Following on from this work, urological cell lines were exposed to urine for up to 12 hours to determine the effect of urine on the ability to classify these cells. Results indicate that urine has no detrimental effect on prediction results.

Zeta potential of motile spores of the green alga Ulva linza and the influence of electrostatic interactions on spore settlement and adhesion strength

The zeta potential of the motile spores of the green alga (seaweed) Ulva linza was quantified by video microscopy in combination with optical tweezers and determined to be −19.3±1.1 mV. The electrostatic component involved in the settlement and adhesion of spores was studied using electret surfaces consisting of PTFE and bearing different net charges. As the surface chemistry remains the same for differently charged surfaces, the experimental results isolate the influence of surface charge and thus electrostatic interactions. Ulva spores were demonstrated to have a reduced tendency to settle on negatively charged surfaces and when they did settle the adhesion strength of settled spores was lower than with neutral or positively charged surfaces. These observations can be ascribed to electrostatic interactions.

Optically trapped probes with nanometer-scale tips for femto-Newton force measurement

We describe the development of a novel force probe, controlled by multiple optical traps, with a nanometer-scale tip that protrudes outside the direct laser radiation field. We have measured forces to an accuracy of 240?fN, which enables future experiments that probe photo-sensitive components (such as biological cells) and non-transparent objects. The probes were produced using two methods, electron beam lithography and two-photon polymerization, with the latter providing approximately twice as much trapping stiffness.

Incandescent porous carbon microspheres to light up cells: solution phenomena and cellular uptake

Carbon based materials are attractive for biological applications because of their excellent biocompatibility profile. Porous carbons with high specific surface area are particularly interesting because it is possible in principle to leverage their properties to deliver high drug payloads. In this work, porous carbon microspheres with high specific surface area were prepared and studied in solution and in cells. Raman optical tweezer trapping of microspheres, excited at 532 nm, results in graphitization and incandescence in solvents that display poor heat conduction. Fluorescence confocal microscopy imaging was used to demonstrate the uptake of fluorescently labelled microspheres by cells and the ability to leverage their optical absorptivity in order to cause carbon graphitization and cell death.

Dynamic position and force measurement for multiple optically trapped particles using a high-speed active pixel sensor

A high frame rate active pixel sensor designed to track the position of up to six optically trapped objects simultaneously within the field of view of a microscope is described. The sensor comprises 520 x 520 pixels from which a flexible arrangement of six independent regions of interest is accessed at a rate of up to 20 kHz, providing the capability to measure motion in multiple micron scale objects to nanometer accuracy. The combined control of both the sensor and optical traps is performed using unique, dedicated electronics (a field programmable gate array). The ability of the sensor to measure the dynamic position and the forces between six optically trapped spheres, down to femtonewton level, is demonstrated paving the way for application in the physical and life sciences.

Optics clustered to output unique solutions: a multi-laser facility for combined single molecule and ensemble microscopy.

Optics clustered to output unique solutions (OCTOPUS) is a microscopy platform that combines single molecule and ensemble imaging methodologies. A novel aspect of OCTOPUS is its laser excitation system, which consists of a central core of interlocked continuous wave and pulsed laser sources, launched into optical fibres and linked via laser combiners. Fibres are plugged into wall-mounted patch panels that reach microscopy end-stations in adjacent rooms. This allows multiple tailor-made combinations of laser colours and time characteristics to be shared by different end-stations minimising the need for laser duplications. This setup brings significant benefits in terms of cost effectiveness, ease of operation, and user safety. The modular nature of OCTOPUS also facilitates the addition of new techniques as required, allowing the use of existing lasers in new microscopes while retaining the ability to run the established parts of the facility. To date, techniques interlinked are multi-photon/multicolour confocal fluorescence lifetime imaging for several modalities of fluorescence resonance energy transfer (FRET) and time-resolved anisotropy, total internal reflection fluorescence, single molecule imaging of single pair FRET, single molecule fluorescence polarisation, particle tracking, and optical tweezers. Here, we use a well-studied system, the epidermal growth factor receptor network, to illustrate how OCTOPUS can aid in the investigation of complex biological phenomena.

The Use of Raman Tweezers and Chemometric Analysis to Discriminate the Urological Cell Lines, PC-3, LNCaP, BPH and MGH-U1

Here we report on investigations into using Raman optical tweezers to analyse both live and chemically fixed prostate and bladder cells. Spectra were subjected to chemometric analysis to discriminate and classify the cell types based on their spectra. Subsequent results revealed the potential of Raman tweezers as a potential clinical diagnostic tool.

Floating micro-structured force probe for 3D imaging and force microscopy on the nanometre scale

A nanometre scale, picoNewton force probe based on advanced optical tweezers control of transparent microstructures will be presented. The probe will be applied to dynamic bio-molecular interactions in solution