Significance: Optical microscopy is characterized by the ability to get high resolution, below 1 μm, high contrast, functional and quantitative images. The use of shaped illumination, such as with lightsheet microscopy, has led to greater three-dimensional isotropic resolution with low phototoxicity. However, in most complex samples and tissues, optical imaging is limited by scattering. Many solutions to this issue have been proposed, from using passive approaches such as Bessel beam illumination to active methods incorporating aberration correction, but making fair comparisons between different approaches has proven to be challenging.
Aim: We present a phase-encoded Monte Carlo radiation transfer algorithm (φMC) capable of comparing the merits of different illumination strategies or predicting the performance of an individual approach.
Approach: We show that φMC is capable of modeling interference phenomena such as Gaussian or Bessel beams and compare the model with experiment.
Results: Using this verified model, we show that, for a sample with homogeneously distributed scatterers, there is no inherent advantage to illuminating a sample with a conical wave (Bessel beam) instead of a spherical wave (Gaussian beam), except for maintaining a greater depth of focus.
Conclusion: φMC is adaptable to any illumination geometry, sample property, or beam type (such as fractal or layered scatterer distribution) and as such provides a powerful predictive tool for optical imaging in thick samples.
Correlation mapping optical coherence tomography (cmOCT) is a powerful technique for the imaging of skin microvessels structure, based on the discrimination of the static and dynamic regions of the tissue. Although the suitability of cmOCT to visualize the microcirculation has been proved in humans and animal models, less evidence has been provided about its application to examine functional dynamics. Therefore, the goal of this research was validating the cmOCT method for the investigation into microvascular function and vasomotion. A spectral domain optical coherence tomography (SD-OCT) device was employed to image 90 sequential three-dimensional (3-D) OCT volumes from the forearm of 12 volunteers during a 25-min postocclusive reactive hyperemia (PORH) test. The volumes were processed using cmOCT to generate blood flow maps at selected cutaneous depths. The maps clearly trace flow variations during the PORH response for both capillaries and arterioles/venules microvascular layers. Continuous blood flow signals were reconstructed from cmOCT maps to study vasomotion by applying wavelet transform spectral analysis, which revealed fluctuations of flow during PORH, reflecting the regulation of microvascular tone mediated by endothelial cells and sympathetic nerves. The results clearly demonstrate that cmOCT allows the generation of functional information that may be used for diagnostic applications.
The cutaneous microcirculation represents an index of the health status of the cardiovascular system. Conventional methods to evaluate skin microvascular function are based on measuring blood flow by laser Doppler in combination with reactive tests such as post-occlusive reactive hyperaemia (PORH). Moreover, the spectral analysis of blood flow signals by continuous wavelet transform (CWT) reveals nonlinear oscillations reflecting the functionality of microvascular biological factors, e.g. endothelial cells (ECs). Correlation mapping optical coherence tomography (cmOCT) has been previously described as an efficient methodology for the morphological visualisation of cutaneous micro-vessels. Here, we show that cmOCT flow maps can also provide information on the functional components of the microcirculation. A spectral domain optical coherence tomography (SD-OCT) imaging system was used to acquire 90 sequential 3D OCT volumes from the forearm of a volunteer, while challenging the micro-vessels with a PORH test. The volumes were sampled in a temporal window of 25 minutes, and were processed by cmOCT to obtain flow maps at different tissue depths. The images clearly show changes of flow in response to the applied stimulus. Furthermore, a blood flow signal was reconstructed from cmOCT maps intensities to investigate the microvascular nonlinear dynamics by CWT. The analysis revealed oscillations changing in response to PORH, associated with the activity of ECs and the sympathetic innervation. The results demonstrate that cmOCT may be potentially used as diagnostic tool for the assessment of microvascular function, with the advantage of also providing spatial resolution and structural information compared to the traditional laser Doppler techniques.
Regenerative medicine has the capability to revolutionise many aspects of medical care, but for it to make the step from
small scale autologous treatments to larger scale allogeneic approaches, robust and scalable label free cell sorting
technologies are needed as part of a cell therapy bioprocessing pipeline. In this proceedings we describe several
strategies for addressing the requirements for high throughput without labeling via: dimensional scaling, rare species
targeting and sorting from a stable state. These three approaches are demonstrated through a combination of optical and
ultrasonic forces. By combining mostly conservative and non-conservative forces from two different modalities it is
possible to reduce the influence of flow velocity on sorting efficiency, hence increasing robustness and scalability. One
such approach can be termed "optically enhanced acoustophoresis" which combines the ability of acoustics to handle
large volumes of analyte with the high specificity of optical sorting.
Lightsheet fluorescence microscopy (LSFM) has rapidly progressed in the past decade from an emerging technology into
an established methodology. This progress has largely been driven by its suitability to developmental biology, where it
is able to give excellent spatial-temporal resolution over relatively large fields of view with good contrast and low
phototoxicity. In many respects it is superseding confocal microscopy. However, it is no magic bullet and still struggles
to image deeply in more highly scattering samples. Many solutions to this challenge have been presented, including,
Airy and Bessel illumination, 2-photon operation and deconvolution techniques. In this work, we show a comparison
between a simple but effective Gaussian beam illumination and Bessel illumination for imaging in chicken embryos.
Whilst Bessel illumination is shown to be of benefit when a greater depth of field is required, it is not possible to see any
benefits for imaging into the highly scattering tissue of the chick embryo.
We report the combined use of optical sorting and acoustic levitation to give particle sorting. Differing sizes of microparticles are sorted optically both with and without the aid of acoustic levitation, and the results compared to show that the use of acoustic trapping can increase sorting efficiency. The use of a transparent ultrasonic transducer is also shown to streamline the integration of optics and acoustics. We also demonstrate the balance of optical radiation pressure and acoustic levitation to achieve vertical sorting.
We experimentally demonstrate continuous attraction of macroscopic targets (> 1 cm) towards the source, against a net momentum flux in the system. Use of a simple setup provides an easily understood illustration of the negative radiation pressure concept for tractor beam, and how these are distinct from the gradient
force acting in conventional optical tweezers. Here, we map out regimes over which
negative radiation forces dominate, and (favorably) compare the thresholds observed to those that emerge from simulations. Theoretical explorations of tractor beam action commonly invoke higher-order Bessel beams, and here we make clear that the reason for this is because of the reduction in axial momentum associated with such hollow-core beams, which allows effects associated with off-axis “skew” momentum to become dominant. Ultimately, there is interest in exploring the language used for describing such effects: radiation pressure versus gradient force (which we suggest might be better described in terms of non‐conservative versus conservative forces), and “orbital” angular momentum (which we suggest might be more appropriately termed “topological” angular momentum).
Whilst the main strength of optical trapping techniques is arguably its precision and dexterity, the complimentary
technique of acoustic trapping offers massive scalability and potentially larger forces. Acoustic traps commonly use
ultrasonic standing waves to trap particles within the nodes of a pressure field, often over distances upwards of a few cm.
Here, an acoustic Bessel beam has been created using a piezoelectric cylinder whereby particles are trapped within the
entire 14 mm-diameter of the transducer (1.5 cm2 trapping area). In optics, Bessel beams have the ability to trap
particles over axial distances of several hundred microns. In this acoustic case, the Bessel function shape of the field is
formed within the entire length of the cylinder (10 mm). Polymer spheres ranging from 1 μm to 100 μm in diameter are
trapped simultaneously within the nodes of the standing wave field, in this case the concentric rings of a Bessel beam.
The smaller particles within this field (< 5m) have also been trapped optically using a single beam optical tweezer, as
the acoustic force scales such that it becomes comparable to that of the optical trap. This allows for a large range of
particle sizes to be simultaneously trapped in a single device, and for large arrays (hundreds of mm2) to be formed
acoustically within which particles can be individually optically trapped. This result demonstrates the complementarity
of optical and acoustic trapping which makes it possible to trap large area arrays of particles whilst retaining the
dexterity to manipulate individual particles.
When samples of interest are small enough, even the relatively weak forces and torques associated with
light can be sufficient for mechanical manipulation, can offer extraordinary position control, and can
measure interactions with three orders of magnitude better resolution than atomic force microscopy.
However, as the components of interest grow to slightly larger length scales (which may yet be of interest
for microfluidic, "lab-on-a-chip" technologies and for research involving biomedical imaging), other
approaches gain strength. This paper includes discussion of the angular momentum carried by sonic beams
that we have implemented both to levitate and controllably rotate disks as large as four inches across.
Discussion of such acoustic beams complements the discussion of the angular momentum carried by light
and, by further analogy, how we view stationary states discussed in quantum mechanics. Hence, a primary
use of the sonic screwdriver is as a model system, although these beams are useful for a variety of other
reasons as well (not least for aberration correction for ultrasonic array systems). Methods, including the use
of holographically structured fields, are discussed.
Here, we present real-space studies of Brownian hard sphere transport though externally defined potential energy
landscapes. Specifically, we examine how colloidal particles are re-routed as moderately dense suspensions pass
through optical lattices, concentrating our attention upon the degree of sorting that occurs in multi-species flows.
While methodologies reported elsewhere for microfluidic sorting of colloidal or biological matter employ active
intervention to identify and selectively re-route particles one-by-one, the sorting described here is passive, with
intrinsically parallel processing. In fact, the densities of co-flowing species examined here are sufficient to allow for
significant many-body effects, which generally reduce the efficiencies of re-routing and sorting. We have studied
four classes of transport phenomena, involving colloidal traffic within, respectively, a static lattice with a DC fluid
flow, a continuously translating lattice with a DC fluid flow, a flashing lattice with AC fluid flow, and a flashing
lattice with combined AC and DC fluid flow. We find that continuous lattice translation helps to reduce nearest
neighbor particle distances, providing promise for efficiency improvements in future high throughput applications.
We present a study into the small particle size and resolution limits of Light Induced Dielectrophoresis (LIDEP). Here the illumination of a photoconductive layer creates virtual electrodes whose associated electric field gradients cause the dielectrophoretic response of the particles. In this way a potential energy landscape can be created that is optically controlled giving reconfigurable control over a large area [1]. In this paper we discuss the interlinked limits of size of particle it is possible to manipulate and the resolution these particles can be manipulated with. We compare traditional dielectrophoresis (DEP) experiments with LIDEP experiments, and discuss the mechanisms behind the physical limits comparing the effects of carrier diffusion verses the spreading of the electric fields in the medium.
The growth of research into microfluidics, especially towards micro-Total Analysis Systems (μTAS), is leading to a demand for highly efficient and accurate methods for analyte delivery, sorting, mixing and analysis. Optical techniques, due to their non-invasive, non-contact properties are ideally suited to integration in to microfluidic systems. One of the key abilities in a μTAS device is the ability to sort microscopic matter. When done optically this typically involves fluorescence detection, management of the information detected and subsequent action such as the actuation of an electric field or electro-mechanical valve. We present here a method whereby the detection of a micro-particle's properties is done passively, with simultaneous separation of those particles. To do this particle streams are injected into a three-dimensional crystal-like lattice of optical intensity maxima. A particle's response to the three-dimensional optical potential landscape formed by the lattice depends on its polarisability. This leads to a sensitivity to size, refractive index and shape. More strongly interacting particles are deflected away from the main flow whilst those that interact weakly are washed straight through the lattice without little or no net deflection. We present analysis of both injection and subsequent re-routing/sorting of particle streams, using body-centred tetragonal and three-dimensional "log-pile" optical lattices to separate both inert colloid and blood cells by refractive index or size. Sorting with an efficiency as high as 96% has been achieved with particle deflections in excess of 45 degrees.
Cells that are exposed to varying amounts of ultrasonic energy in the presence of ultrasound contrast agent (UCA) may undergo either permanent cell membrane damage (lethal sonoporation), or a transient enhancement of membrane permeability (reversible or non lethal sonoporation). The merits of each mode are clear; lethal sonoporation constitutes a significant tumour therapy weapon, whilst its less intrusive counterpart, reversible sonoporation, represents an effective non-invasive targeted drug delivery technique.
Our working hypothesis for understanding this problem was that the root cause and effect in sonoporation involves the interaction of individual cells with single microbubbles, and to that end we devised an experiment that facilitates video rate observation of this specific scenario under well defined optical control. Specifically, we have constructed an innovative hybridization apparatus involving holographic optical trapping of single and multiple UCA microbubbles, together with the facility to irradiate with MHz pulsed ultrasound energy in the presence cancerous cells. This approach allows the isolation of a target microbubble from a resident population and the relocation to a [controllable] predetermined position relative to a cell within a monolayer. Frame extraction from standard framing rate video microscopy demonstrates the individuality of single microbubble-cell interactions. We describe a fluorescence microscopy protocol that will allow future study of the potential to deliver molecular species to cells, the dependence of the delivery on the initial microbubble-cell distance and to determine the targeted cell survival.
The analogous nature of dislocations in crystals and light leads to some interesting links between the properties of optical and crystal dislocations. A dislocation in a crystal can be described by a Burgers vector b, whilst a dislocation in a laser mode such as the vortex in a Laguerre Gaussian beam, can be described by a topological charge l2. By illuminating both optically trapped and self-assembled two-dimensional colloidal crystals with a Gaussian laser beam, we show a direct link between crystal and light dislocations, where the first order diffraction pattern from a crystal with Burgers' vector b = na contains vortex laser modes of topological charge l=±mn, (where n is an integer, a is the lattice constant of the crystal and m is an integer corresponding to the diffraction order).
In the emergent field of microfluidics there is the desire to process ever decreasing amounts of analyte. This will challenge our ability to pump, move and mix small volumes of fluid and will require the controlled actuation of various microcomponents. A valuable tool for micromanipulation is optical tweezing. Here the forces due to the reflection and refraction of light at a dielectric interface can be used to pick up, move and rotate microscopic particles. A powerful method of rotating microparticles at high rates in optical tweezers is the use of birefringence. A birefringent particle can be rotated in a standard optical trap simply by manipulating the input light's state of polarisation, however this requires specialist materials that are not easily micromachined. Here we present the modelling, fabrication and rotation of form birefringent microgears. The birefringence comes from the shape of the microgear (a 1D photonic crystal etched into the center of the microgear) rather than the materials intrinsic properties allowing us to use materials convenient for microfabrication.
We have demonstrated the orientation of microgears with the direction of polarisation of linearly polarised light and rotation by rotating the direction of polarisation of linearly polarised light and by illumination with circularly polarised light. We have modelled and experimentally measured the magnitude of the birefringence with good agreement. We have also demonstrated that one microgear can be used to rotate a second optically trapped microgear clearly demonstrating how form birefringent microgears could be used to actuate a micromachine such as a microgear pump.
The path that a mesoscopic polarisable particle takes as it flows through a lattice of intensity maxima and minima (optical lattice) depends crucially upon the degree to which it interacts with the lattice. Two particles of dissimilar size, refractive index or even shape will interact in a different manner with such a lattice. Combining this selective interaction with a guiding mechanism has allowed us to achieve lateral separation of particles by all these properties simply by flowing them through an angled optical lattice. We present such particle separation in a variety of three-dimensional optical lattices discussing the importance of parameters such as flow speed, lattice intensity, lattice constant, lattice angle, maxima interconnectivity and flow chamber design. We also present cell sorting with the separation of erythrocytes from lymphocytes and present our flow chamber fabrication methods.
Laguerre-Gaussian (LG) laser modes (annular shaped modes with helical phase fronts) are used to both manipulate and cut microscopic particles. We use holographically produced LG laser modes to manipulate microscopic bubbles. Interference patterns formed from LG modes of opposite phase helicity are used to create 3D structures and to continuously rotate glass rods. The technique of using and LG beam to create microscopic sections of chromosomes is described.
We demonstrate the use of the angular Doppler effect to obtain continuous motion of interference patterns. A small frequency shift between two beams can create such a moving pattern. By rotating a half wave plate in one arm of an interferometer, frequency shifts in the optical domain from less than 1 Hertz to kHz are achieved. We apply moving interference patterns in an optical tweezers set-up to enable controlled and continuous motion of optically trapped particles and structures.
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