Graeme Whyte

Heriot-Watt University · Institute of Biological Chemistry, Biophysics and Bioengineering

The recent boom in single-cell omics has brought researchers one step closer to understanding the biological mechanisms associated with cell heterogeneity. Rare cells that have historically been obscured by bulk measurement techniques are being studied by single cell analysis and providing valuable insight into cell function. To support this progre...

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of...

Micron-scale droplets isolated by an immiscible liquid can provide miniaturised reaction vessels which can be manipulated in microfluidic networks, and has seen a rapid growth in development. In many experiments, the precise volume of these microdroplets is a critical parameter which can be influenced by many external factors. In this work, we demo...

There is mounting evidence that the nuclear envelope, and particularly the lamina, plays a critical role in the mechanical and regulation properties of the cell and changes to the lamina can have implications for the physical properties of the whole cell. In this study we demonstrate that the optical stretcher can measure changes in the time-depend...

We describe a quantitative, high-precision, high-throughput method for measuring the mechanical properties of cells in suspension with a microfluidic device, and for relating cell mechanical responses to protein expression levels. Using a high-speed (750 fps) charge-coupled device camera, we measure the driving pressure Δp, maximum cell deformation...

The prevalence of clinically-relevant bacterial strains resistant to current antibiotic therapies is increasing and has been recognized as a major health threat. For example, multidrug-resistant tuberculosis and methicillin-resistant Staphylococcus aureus are of global concern. Novel methodologies are needed to identify new targets or novel compoun...

Microfluidic microdroplets have increasingly found application in biomolecular sensing as well as nanomaterials growth. More recently the synthesis of plasmonic nanostructures in microdroplets has led to surface-enhanced Raman spectroscopy (SERS)-based sensing applications. However, the study of nanoassembly in microdroplets has previously been hin...

We describe a method for quantifying the mechanical properties of cells in suspension with a microfluidic device consisting of a parallel array of micron-sized constrictions. Using a high-speed CCD camera, we measure the speed, deformation and entry time into microconstrictions of several hundred cells per minute. Cell entry time Δt into microconst...

We describe a method for quantifying the mechanical properties of cells in suspension with a microfluidic device consisting of a parallel array of micron-sized constrictions. Using a high-speed charge-coupled device camera, we measure the flow speed, cell deformation, and entry time into the constrictions of several hundred cells per minute during...

Phospholipid vesicles are common model systems for cell membranes. Important aspects of the membrane function relate to its mechanical properties. Here we have investigated the deformation behaviour of phospholipid vesicles in a dual-beam laser trap, also called an optical stretcher. This study explicitly makes use of the inherent heating present i...

Flow cytometry provides a high throughput, multi-dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow,...

The mechanical properties of biological cells have long been considered as inherent marker of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence d...

The classical purpose of optical fibres is delivery of either optical power, as for welding, or temporal information, as for telecommunication. Maximum performance in both cases is provided by the use of single-mode optical fibres. However, transmitting spatial information, which necessitates higher-order modes, is difficult because their dispersio...

Determining cell mechanical properties is increasingly recognized as a marker-free way to characterize and separate biological cells. This emerging realization has led to the development of a plethora of appropriate measurement techniques. Here, we use a fairly novel approach, deterministic lateral displacement (DLD), to separate blood cells based...

This invention relates to microfluidic systems and more particularly to methods and apparatus for accessing the contents of micro droplets (114) in an emulsion stream. A method of accessing the contents of a droplet (114) of an emulsion in a microfluidic system, the method comprising: flowing the emulsion alongside a continuous, non-emulsive stream...

In this paper we describe a pneumatically actuated fibre-optic spanner integrated into a microfluidic Lab-on-a-Chip device for the controlled trapping and rotation of living cells. The dynamic nature of the system allows interactive control over the rotation speed with the same optical power. The use of a multi-layer device makes it possible to rot...

A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe ce...

The combination of optics and microfluidics is a very powerful and non invasive tool to perform single cell analysis and imaging. Being able to measure properties at a single cell level is of fundamental importance to understanding cellular systems and their heterogeneity. We present flow cytometry systems with added rotation techniques to gain iso...

We use a dual-beam fibre trap integrated into a microfluidic system to hold single cells and rotate them about an axis perpendicular to the optical axis to give an isotropic tomographic image of the cell

The theory for the deformations of a spheroidal particle is of great scientific interest in numerous physical and biological problems ranging from fracture analysis of plain solids to the compression of biological cells in an atomic force microscope or during micropipette aspiration. Using a formulation in terms of Papkovich–Neuber potentials, we d...

We have generated a series of stable cell lines expressing fluorescently tagged nuclear-envelope proteins for the investigation of the nuclear contribution to cell mechanics

We present two electromagnetic frameworks to compare the surface stresses on spheroidal particles in the optical stretcher (a dual-beam laser trap that can be used to capture and deform biological cells). The first model is based on geometrical optics (GO) and limited in its applicability to particles that are much greater than the incident wavelen...

We present a new class of integrated optical devices, fabricated by femtosecond laser micromachining, that allows for mechanical probing, fluorescence detection and sorting of single cells by means of optical forces inside a microfluidic chip.

The combination of high power laser beams with microfluidic delivery of cells is at the heart of high-throughput, single-cell analysis and disease diagnosis with an optical stretcher. So far, the challenges arising from this combination have been addressed by externally aligning optical fibres with microfluidic glass capillaries, which has a limite...

Although cellular mechanical properties are known to alter during stem cell differentiation, understanding of the functional relevance of such alterations is incomplete. Here, we show that during the course of differentiation of human myeloid precursor cells into three different lineages, the cells alter their viscoelastic properties, measured usin...

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Nuclear morphology as further proof of differentiation and cell viability during differentiation. A. Representative confocal images (single slices) of cells stained with a green fluorescent cytoplasmic dye (Mitotracker Orange) and a red fluorescent nuclear dye (Syto 61). Neutrophils have lobulated nuclei (bands and segmented), usually 3–5 lobes but...

Distribution of creep compliance and stress- as well as set-up independence of lineage specificity in creep compliance. A. The histograms show the distribution of peak compliance (at t = 4 s) for the HL60 and all differentiated lineages plotted in Fig. 1C. Although the populations overlap there are significant shifts in compliance (decrease to the...

Short time scale advection of monocytes through microfluidic channels. Monocytes need much less time than the macrophages in Video S1 to slip through the same channels and at the same driving pressure. Video presented in real time. (WMV)

Size changes during the differentiation of cells over several days. Macrophages become larger with differentiation, neutrophils and monocytes become smaller. The cell radii shown (mean ± SEM) were obtained during the OS measurements reported in Fig. 2 (with the same number of cells in each measurement) and used for the proper normalization of the s...

Conversion of compliance to complex modulus using power law and mechanical models within quasi-linear viscoelastic regimes. A. Two approximately linear viscoelastic regimes for cell line model: the first ending around 0.97 Pa, corresponds to about 0.75 W per fibre, thus, engendering the experiments at 0.7 W per fibre (all experiments reported in th...

F-actin depolymerization reduces transition times in all cell types. The transition time dropped to less than 0.8 s in all cell types treated with 2 µM cytochalasin D (cytoD). (TIF)

Primary stem cells (CD34+) are less compliant than primary monocytes and have highly reproducible viscoelastic properties across different healthy donors. A. CD34+SCs are less compliant than primary monocytes. B. Box plots for the results of A, showing highly significant difference (p<0.0001). C. Primary CD34+ stem cells from different donors show...

Macrophages derived from primary stem cells (CD34+) are less compliant than undifferentiated CD34+ stem cells. A. Upon differentiation, CD34+ derived macrophages (CD34+ pMac, n = 32) become less compliant than undifferentiated CD34+SCs (n = 45). B. Box plots for the results of A, showing significant difference (p<0.01). (TIF)

Advection times at 10 mbar. HL60 cells (n = 117) required a significantly longer (p<0.001) advection time of 0.43±0.07 s to go through the 10×12 µm channel compared to 0.13±0.03 s for monocytes (n = 88) and 0.15±0.04 s neutrophils (n = 49). (TIF)

Short time scale advection of macrophages through microfluidic channels. Macrophages obviously need quite some time to deform before they can slip through. Video presented in real time. (WMV)

Myelination and its regenerative counterpart remyelination represent one of the most complex cell-cell interactions in the central nervous system (CNS). The biochemical regulation of axon myelination via the proliferation, migration, and differentiation of oligodendrocyte progenitor cells (OPCs) has been characterized extensively. However, most bio...

Myelination, the process in which myelin sheaths are formed around axons, is fundamental for development and regeneration of central neural system (CNS). Critical to this process is the differentiation of oligodendrocyte precursor cells (OPCs), which is concurrent with gross morphological changes as the cells migrate to and wrap around axons. Studi...

In this work we present data showing deformability-based cell separation in a deterministic lateral displacement (DLD) device. We use cells of defined stiffness (glutaraldehyde cross-linked erythrocytes) to test the performance of the device across a range of cell stiffness and applied shear rates. Optical stretching is used as an independent metho...

Optical traps are nowadays quite ubiquitous in biophysical and biological studies. The term is often used synonymously with optical tweezers, one particular incarnation of optical traps. However, there is another kind of optical trap consisting of two non-focused, counter-propagating laser beams. This dual-beam trap predates optical tweezers by alm...

There has been an increasing drive toward miniaturizing and accelerating experiments with droplet-based microfluidics across the chemical disciplines. Current applications take advantage of the numerous techniques for manipulating nano- to femtoliter droplets within microfluidic devices. To expand the range of possible applications, we have develop...

Using microfluidic techniques and a novel fluorous-tagged palladium catalyst, we generated droplet reactors with catalytically active walls and used these compartments for small molecule synthesis.

A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluores...

Fully integrated: Mass spectrometry has been integrated into a detection scheme for microdroplets that are created within microfluidic channels (see picture, scale bar 200 microm). This technique allows droplets to be identified based on the compounds they contain, and combines fluorescence screening with MS analysis. These experiments indicate how...

We describe the design, fabrication and use of a single-layered poly(dimethylsiloxane) microfluidic structure for the entrapment and release of microdroplets in an array format controlled entirely by liquid flow. Aqueous picoliter droplets are trapped en masse and optically monitored for extended periods of time. Such an array-based approach is use...

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of vario...

We present a versatile method for selective mode coupling into higher-order modes of photonic crystal fibers, using holograms electronically generated by a spatial light modulator. The method enables non-mechanical and completely repeatable changes in the coupling conditions. We have excited higher order modes up to LP31 in hollow-core photonic cry...

We investigate the capability of open source freeware ray tracing rendering software to demonstrate the imaging properties of canonical optical resonators. This software can directly visualize the imaging properties of canonical resonators. We can also demonstrate effects such as the trapping of light rays in geometrically stable resonators and the...

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitori...

The size of bright structures in traveling-wave light fields is limited by diffraction. This in turn limits a number of technologies, for example, optical trapping. One way to beat the diffraction limit is to use evanescent waves instead of traveling waves. Here we apply a holographic algorithm, direct search, to the shaping of complex evanescent-w...

We report a holographic assembler workstation for optical trapping and micro-manipulation. The workstation is based on a titanium sapphire laser, making it particularly suited for biomaterials and incorporates a choice of user interfaces for different applications. The system is designed around a commercial inverted microscope and is configured suc...

Microdroplets have great potential for high-throughput biochemical screening. We report the design of an integrated microfluidic device for droplet formation, incubation and screening. Picolitre water-in-oil droplets can be stored in a reservoir that contains approximately 10(6) droplets. In this reservoir droplets are stable for at least 6 h, whic...

Der Inhalt von Mikrotröpfchen kann auf einem Chip extrahiert und mithilfe von Elektrokoaleszenz in einen kontinuierlichen Mikrofluidstrom überführt werden. Einzelne Tröpfchen lassen sich nach ihrem Inhalt auswählen. In einem Modellversuch wurde ein Fluoreszenzdetektionssystem genutzt, um einzelne fluoreszierende Kügelchen zu erkennen oder Tröpfchen...

Optical tweezers manipulate microscopic particles using foci of light beams. Their performance is therefore limited by diffraction. Using computer simulations of a model system, we investigate the application of superresolution holography for two-dimensional (2D) light shaping in optical tweezers, which can beat the diffraction limit. We use the di...

The contents of microdroplets can be extracted on-chip and incorporated by electrocoalescence into a continuous microfluidic stream. Individual droplets can be selected based on their contents. As a proof of principle, a fluorescence detection system was used to collect droplets containing low levels of a fluorescent dye (see picture of droplets pa...

Holograms can shape the 3-D intensity distribution of light beams. Here we describe work on 3-D intensity shaping of various kinds of beams, including monochromatic travelling waves, evanescent waves and self-reconstructing beams.

This paper investigates the use of holographic algorithms to create more complex evanescent-wave fields. The authors have written a direct-search/simulated-annealing algorithm for shaping the 3D intensity pattern of evanescent wave fields. This direct-search algorithm includes a forward model that calculates the 3D intensity pattern due to a given...

We report the development of a joystick controlled gripper for the real-time manipulation of micron-sized objects, driven using holographic optical tweezers (HOTs). The gripper consists of an arrangement of four silica beads, located in optical traps, which can be positioned and scaled in order to trap an object indirectly. The joystick can be used...

We have developed a real time interface for holographic optical tweezers where the operator’s fingertips are mapped to the positions of silica beads captured in optical traps. The beads act as the fingertips of a microhand which can be used to manipulate objects that otherwise do not lend themselves to tweezers control, e.g. objects that are strong...

We investigate the property of isotropic harmonic traps to Fourier transform a weakly interacting Bose-Einstein Condensate (BEC) every quarter of a trap period. We solve the Gross-Pitaevskii equation numerically to investigate the time evolution of interacting BECs in the context of the Fourier transform, and we suggest potential applications.

Optical tweezers use the electric-field gradient-force associated with tightly focused laser beams to trap micron-sized objects at the beam focus. Over the last few years optical tweezers have been revolutionized by the addition of spatial light modulators to split the laser beam into many traps that can be individually controlled; a technique call...

When light is transmitted along the axis of a rotating glass rod, the polarization of the light is rotated through a small angle [Proc. R. Soc. London, Ser. A349, 423 (1976)]. Under the same conditions, we predict a rotation of the transmitted image by exactly the same angle. The treatment of the two effects in terms of light's spin and orbital ang...

We have developed iterative algorithms for the calculation of holograms for non-diffracting or self-imaging light beams. Our methods make use of the special Fourier-space properties of the target beams. We demonstrate experimentally the holographic generation of perhaps the most challenging type of beam: a self-imaging beam shaped in more than one...

Light shaping is usually concerned with shaping the bright part of the beam. Here we investigate a technique for shaping a light beam's vortex lines, which are completely dark.

We use a three-dimensional Gerchberg–Saxton algorithm (Shabtay (2003) Opt. Commun. 226 33) to calculate the Fourier-space representation of physically realizable light beams with arbitrarily shaped three-dimensional intensity distributions. From this representation we extract a phase-hologram pattern that allows us to create such light beams experi...

We have designed interferometers that sort Bose-Einstein condensates into their vortex components. The Bose-Einstein condensates in the two arms of the interferometer are rotated with respect to each other through fixed angles; different vortex components then exit the interferometer in different directions. The method we use to rotate the Bose-Ein...

We examine the Bose-Einstein-condensate (BEC) equivalent of transverse aspects of laser resonators. We model numerically repeated focussing of a 2-dimensional BEC, which could be achieved in practice by a series of far off-resonant light pulses. We show for a range of non-linear coefficients that such a series of light pulses traps the BEC. We also...

Thesis (Ph.D.) - University of Glasgow, 2007. Includes bibliographical references.