Our recent THz imaging system performs full-frame high-speed imaging (12000 fps) by exploiting efficient THz-to-optical conversion in an excited Caesium atomic vapour.
Structured Illumination Microscopy (SIM) has revolutionized optical microscopy pushing beyond the diffraction limit. At THz frequencies the diffraction limit is measured on the sub-mm scale therefore would benefit from improvement.
Implementing Structured Illumination Super Resolution at the THz regime has previously been unattractive due to the long acquisition times of conventional THz detectors, compounded by the requirement of multiple images for super-resolution image reconstruction.
Using our high-speed THz imaging system, we investigate the application of Structured Illumination Super Resolution Imaging as a method to improve spatial resolution, while maintaining the high penetrating properties and high detection sensitivity at 0.55THz.
SignificancePostoperative surgical wound infection is a serious problem around the globe, including in countries with advanced healthcare systems, and a method for early detection of infection is urgently required.AimWe explore spatial frequency domain imaging (SFDI) for distinguishing changes in surgical wound healing based on the tissue scattering properties and surgical wound width measurements.ApproachA comprehensive numerical method is developed by applying a three-dimensional Monte Carlo simulation to a vertical heterogeneous wound model. The Monte Carlo simulation results are validated using resin phantom imaging experiments.ResultsWe report on the SFDI lateral resolution with varying reduced scattering value and wound width and discuss the partial volume effect at the sharp vertical boundaries present in a surgical incision. The detection sensitivity of this method is dependent on spatial frequency, wound reduced scattering coefficient, and wound width.ConclusionsWe provide guidelines for future SFDI instrument design and explanation for the expected error in SFDI measurements.
Surgical site infection is a significant threat to the patient after surgery and is associated with over a third of postoperative deaths. In this work we evaluate spatial frequency domain imaging (SFDI) for clinical use in monitoring wound structural changes and early signs of infection. We use both Monte Carlo and experimental techniques to image embedded vertical wound models. The simulation aids in analyzing heterogeneity between tissues with different optical parameters to detect changes due to infection. The exact resolution of SFDI measurement for scattering and wound width will be discussed.
To unlock the massive economic and clinical potential of the biophotonics research field several barriers to market must be overcome. The National Centre for Healthcare Photonics, set up in 2018 at NETPark, UK, is an £18 million centre dedicated to supporting companies of all sizes in translating research into commercial products. This presentation will detail case studies demonstrating effective partnerships between local companies, non-governmental organisations and universities to bring healthcare products utilising photonics to market more effectively.
A dedicated project, Spotlight, funded by the ERDF, provides healthcare photonics SMEs and start-ups with funded support including staff time from Durham University and the Centre for Process Innovation (CPI), an organisation specialising in supporting the development of next-generation manufacturing organisations. Examples of support that Spotlight offers include:
- access to lab space and state-of-the-art research facilities;
- proof-of-concept research;
- system design, prototyping and validation activity;
- manufacture of equipment for clinical investigation;
- regulatory compliance support;
- health economics modelling
- commercialisation support;
- pathways to generating clinical evidence.
Typically, the SMEs that partner with Spotlight have expertise in several areas and research fields but lack either photonics expertise or access to photonics equipment and do not have the scale or resources to obtain these feasibly. We will present examples of SMEs that have received assistance from Spotlight to enable commercial translation of research. Examples are taken at different stages of product development and different biophotonics technologies and demonstrate the success of interdisciplinary academic-industry partnerships in translating research to market.
We present an achromatic confocal laser scanning system capable of recording spectrally resolved fluorescence lifetime images (sFLIMs) at a rate of >8 frames per second (FPS) for a 128 x 128 image. This frame rate was achieved by optimizing the processing of lifetime calculations from previous results which demonstrated >4 FPS sFLIM imaging. The imaging system is achromatic for a spectral range of 400 - 900 nm, achieved by using reflective optics instead of a transmissive lens system, except for the primary objectives. Two excitation sources have been integrated into the system, 485 nm and 640 nm laser diodes with a pulse width of <70 ps and <90 ps respectively. Imaging is performed via a galvanometric mirror system which scans the laser beam over the sample with the ability to change the Field of View (FOV) on the fly. The collected fluorescence signal is focused into a multimode fiber via a second objective and recollimated onto a transmissive grating for spectral dispersion onto a novel complementary metal–oxide–semiconductor single photon avalanche diode (CMOS SPAD) line array sensor. This sensor can perform lifetime histogram generation on-chip and process over 16.5 Giga events/s enabling fast lifetime data acquisition. High speed sFLIM is demonstrated through imaging of convallaria majalis sections.
The vascular endothelium is a complex single layered network of cells which signal via the release of Ca2+ ions; the study of endothelial cell function and interactions in response to stimuli provides useful information for medical research into, for example, hypertension, diabetes and heart failure. A side viewing GRIN imaging system has previously been used to view calcium signalling in the endothelium [1] utilising a low numerical aperture GRIN rod and microscope objective to increase the imaging depth and image a large number of cells over the curved inner artery surface. This allows cells to be imaged in near-physiological conditions, as opposed to imaging of flattened arteries; however, the use of GRIN lenses introduces optical aberrations. Resolution is also limited from using a low numerical aperture system.
In this work we investigate this important imaging challenge further with a view to compensating for both the cylindrically-curved geometry of the arteries and field-aberrations present in the optical system. The field aberrations in the imaging optics and resulting from the curved surface/planar sensor mismatch are quantified to allow for corrections to be made through introducing field curvature and aberration correction into the imaging path. This new instrumentation opens up the potential to image calcium signalling within large numbers of cells to try and understand the complex patterns which are produced in response to a range of stimuli.
In this research we outline the optical background and manufacture of the OPTA device: an Optical Probe to evaluate Tissue Atrophy. We present a portable profiling system capable of directly interrogating the integrity of human skin in vivo. The device utilizes laser speckle and spectral imaging modalities to measure a suite of physical parameters to aid clinical assessment, in particular surface roughness and blood oxygenation. We provide a detailed discussion on the relation between laser speckle scattering and the influence of surface texture. This leads us to discuss how these measurements can relate a participant’s perception of skin pathologies in vivo and discuss the potential clinical application. We will first discuss the technical information relating to the design and assembly of the Optical Probe to evaluate Tissue Atrophy (OPTA) probe, utilizing a biocompatible 3D printing material, polymer optical fibers and custom miniature cage system optics. Finally, we will present the practical use of this device using our results of a pilot study to trial the OPTA device, in house.
Recent advances in optical microscopy, in particular those based on light sheet imaging, have significantly advanced the field of long term in vivo imaging with minimal perturbation to the sample. This means that there is now an interest in studying the processes by which a body repairs damage, in particular during development. A genetically encoded protein, KillerRed, is now available that is phototoxic, killing the cells in which it is present when illuminated with light at 561nm. The presentation will report for the first time its use in zebrafish with localised cell ablation in a SPIM system.
We report on ablation within the living sample either through the use of the light sheet within the microscope, but more precisely using a beam introduced through the imaging arm of a SPIM microscope controlled by adaptive optics to ensure localization of the activation. The beam profile is optimized to target individual cells within the kidney of the fish and the loss of fluorescence from the KillerRed is used to quantify the damage. The integration of the adaptive optics and opto-genetic encoding and activation of the KillerRed will be demonstrated to have the ability to ablate single cells deep within a living zebrafish with 100% survival of the fish. The presentation will illustrate how significant advances in the life sciences can be made through multidisciplinary research with optical expertise.
All blood vessels are lined with a single layer of endothelia cells which play a vital role in controlling the vessels in terms of blood flow, angiogenesis, vascular remodelling, response to pressure changes and blood borne chemical markers. Traditionally such responses to stimuli are examined in isolated cells, in a vascular preparation in which the vessels are opened up to lie flat or using relatively slow confocal or non-linear microscopy from the outside. We have developed a miniature probe that enables high speed, widefield imaging from within an intact pressurised vessel.
This presentation will discuss the development of a 750 micron diameter probe which views orthogonally to the main optical axis to provide sub-cellular resolution images of around 300 cells in an intact, curved artery with the ability to rapidly change focus. Results of the imaging performance will be presented along with the biological context illustrating how this novel imaging modality when coupled with computational modelling enabled a new insight into the biological signalling processes within an intact vessel. By combining this new imaging system with a novel image processing pipeline results will be presented illustrating that the response to certain agonists is affected by pressure and the changing shape of the cells controls this response during a pressure rise. The work illustrates the way that an interdisciplinary approach bringing together novel optics, image processing, computational simulation and biology can lead to insights in the life sciences.
A Telescope window is a novel concept of transformation-optics consisting of an array of micro-telescopes, in our configuration, of a Galilean type. When the array is considered as one multifaceted device, it acts as a traditional Galilean telescope with distinctive and attractive properties such as compactness and modularity. Each lenslet, can in principle, be independently designed for a specific optical function. In this paper, we report on the design, manufacture and prototyping, by diamond precision machining, of 2 concepts of telescope windows, and discuss both their performances and limitations with a view to use them as potential low vision aid devices to support patients with macular degeneration.
Pixellated Optics, a class of optical devices which preserve phase front continuity only over small sub areas of the device, allow for a range of uses that would not otherwise be possible. One potential use is as Low Vision Aids (LVAs), where they are hoped to combine the function and performance of existing devices with the size and comfort of conventional eyewear. For these devices a Generalised Confocal Lenslet Array (GCLA) is designed to magnify object space, creating the effect of traditional refracting telescope within a thin, planar device. By creating a device that is appreciably thinner than existing LVA telescopes it is hoped that the comfort for the wearer will be increased. We have developed a series of prototype GLCA-based devices to examine their real-world performance, focussing on the resolution, magnification and clarity of image attainable through the devices. It is hoped that these will form the basis for a future LVA devices. This development has required novel manufacturing techniques and a phased development approach centred on maximising performance. Presented here will be an overview of the development so far, alongside the performance of the latest devices.
We present two applications of spectroscopy to help in the development and production of fast moving consumer goods.
We have developed an instrument which combines time-gated Raman spectroscopy integrated with a fiber optic probe bundle for multi-spectral, multi-point investigation of the distribution of chemicals in complex powder mixtures by separation of their individual Raman spectra. The combined instrumentation design is designed for application in a production environment. This finds particular utility in monitoring the production and potential segregation of washing powders, which require consistency; particularly in the developing world where the efficiency of washing with small amounts of powder is beneficial. However, washing powders, in line with many powder products, have the additional problem of an overwhelming fluorescence signal which is stronger than the Raman signal, arising from the use of artificial whiteners added to such powders. We overcome this through the use of a novel time-gating method separate the “instantaneous Raman signal” from the time delayed fluorescence emission.
We will also present a novel use of confocal microscopy in obtaining high resolution images of fluorescently labelled mascara on eyelashes. This is achieved by mixing mascara with a small amount of fluorescein powder. From these images it is possible to see phenomena such as the bridging of mascara between lashes and to accurately determine the thickness of the mascara. This technique has potential in the testing of mascara by cosmetic companies, and also in monitoring other reactions which involve a waxy substrate which adheres to a curved surface.
The two methods demonstrate how significant commercial challenges can be solved through the application of methods more associated with academic research.
We demonstrate a fast two-color widefield fluorescence microendoscopy system capable of simultaneously detecting several disease targets in intact human ex vivo lung tissue. We characterize the system for light throughput from the excitation light emitting diodes, fluorescence collection efficiency, and chromatic focal shifts. We demonstrate the effectiveness of the instrument by imaging bacteria (Pseudomonas aeruginosa) in ex vivo human lung tissue. We describe a mechanism of bacterial detection through the fiber bundle that uses blinking effects of bacteria as they move in front of the fiber core providing detection of objects smaller than the fiber core and cladding (∼3 μm). This effectively increases the measured spatial resolution of 4 μm. We show simultaneous imaging of neutrophils, monocytes, and fungus (Aspergillus fumigatus) in ex vivo human lung tissue. The instrument has 10 nM and 50 nM sensitivity for fluorescein and Cy5 solutions, respectively. Lung tissue autofluorescence remains visible at up to 200 fps camera acquisition rate. The optical system lends itself to clinical translation due to high-fluorescence sensitivity, simplicity, and the ability to multiplex several pathological molecular imaging targets simultaneously.
Imaging applications increasingly require light sources with high spectral density (power over spectral bandwidth. This has led in many cases to the replacement of conventional thermal light sources with bright light-emitting diodes (LEDs), lasers and superluminescent diodes. Although lasers and superluminescent diodes appear to be ideal light sources due to their narrow bandwidth and power, however, in the case of full-field imaging, their spatial coherence leads to coherent artefacts, such as speckle, that corrupt the image. LEDs, in contrast, have lower spatial coherence and thus seem the natural choice, but they have low spectral density. Random Lasers are an unconventional type of laser that can be engineered to provide low spatial coherence with high spectral density. These characteristics makes them potential sources for biological imaging applications where specific absorption and reflection are the characteristics required for state of the art imaging. In this work, a Random Laser (RL) is used to demonstrate speckle-free full-field imaging for polarization-dependent imaging in an epi-illumination configuration. We compare LED and RL illumination analysing the resulting images demonstrating that the RL illumination produces an imaging system with higher performance (image quality and spectral density) than that provided by LEDs.
Using compact and affordable instrumentation based upon fluorescent confocal imaging we have tracked the movement
of autofluorescent compounds through skin in near real time with high temporal and spatial resolution and sensitivity.
The ability to measure the diffusion of compounds through skin with such resolution plays an important role for
applications such as monitoring the penetration of pharmaceuticals applied to skin and assessing the integrity of the skin
barrier. Several measurement methods exist, but they suffer from a number of problems such as being slow, expensive,
non-portable and lacking sensitivity. To address these issues, we adapted a technique that we previously developed for
tracking fluorescent compounds in the eye to measure the autofluorescence and the diffusion of externally applied
fluorescent compounds in skin in vivo. Results are presented that show the change in autofluorescence of the volar
forearm over the course of a week. We furthermore demonstrate the ability of the instrument to measure the diffusion
speed and depth of externally applied fluorescent compounds both in healthy skin and after the skin barrier function has
been perturbed. The instrument is currently being developed further for increased sensitivity and multi-wavelength
excitation. We believe that the presented instrument is suitable for a large number of applications in fields such as
assessment of damage to the skin barrier, development of topical and systemic medication and tracking the diffusion of
fluorescent compounds through skin constructs as well as monitoring effects of skin products and general consumer
products which may come into contact with the skin.
Adaptive optics (AO) can potentially allow high resolution imaging deep inside living tissue, mitigating against the loss of resolution due to aberrations caused by overlying tissue. Closed-loop AO correction is particularly attractive for moving tissue and spatially varying aberrations, but this requires direct wavefront sensing, which in turn requires suitable "guide stars" for use as wavefront references. We present a novel method for generating an orthogonally illuminated guide star suitable for direct wavefront sensing in a wide range of fluorescent biological structures, along with results demonstrating its use for measuring time-varying aberrations, in vivo.
Wide-field fluorescence microscope techniques such as single/selective plane illumination microscope (SPIM) are typically configured to image large regions of a sample at once. Here the illumination beam provides uniform excitation of several biological features across the region, `sliced' to a thickness of between 5-10 microns. In this paper we propose a simple alteration to the optical configuration of a SPIM by switching the light-sheet- forming cylindrical lens with a spatial light modulator. This has the potential to adaptively reconfigure the light sheet geometry to improve the optical sectioning of specific biological features, rather than the thicker sectioning of several features at once across a larger observation field-of-view. We present a prototype version of such a system, referred to as an Adaptive-SPIM (A-SPIM) system. We then suggest that the direct recording of illumination beam shapes within the working microscope system can better facilitate the analysis and subsequent re-configuration of the illumination beam to a specific geometry, and summarise the design and operation of a device that we have developed specifically for this purpose. We finally present reconstructed quantitative three dimensional flux maps of illumination beams from three microscope configurations taken using this miniature high-dynamic range beam profiling device, comparing the beam geometry of a regular SPIM system with our prototype A-SPIM system, and suggesting future improvements.
Selective plane illumination microscopy (SPIM) is a 3D imaging technique that uses a sheet of light to optically section a sample in vivo. A cylindrical lens focuses collimated light in one dimension, producing a sheet that is formed in the sample via an objective lens. Any optical power within the sample will additionally refract the light sheet passing through it. We exploit this effect to track the development of the optical power of the zebrafish lens over the first 4 days post fertilisation (dpf). We show that light is focussed on to the photoreceptor layer of the retina at 4 dpf.
The surface of the skin plays an important role in the diagnosis of many clinical conditions, in relation to ageing and the acceptance of many consumer products. Considerable resource has been applied to skin in terms of cancer diagnosis but the actual surface finish of the tissue has been frequently overlooked. An optical system and associated imaging processing method has been developed which analyses the speckle pattern, recorded on a basic digital imaging system, and provides a quantitative analysis of the surface roughness. Results demonstrate that these measurements can be linked with more qualitative perceptions of skin quality (roughness).
Our sight is a major contributor to our quality of life. The treatment of diseases like macular degeneration and glaucoma, however, presents a challenge as the delivery of medication to ocular tissue is not well understood. The instrument described here will help quantify targeted delivery by non-invasively and simultaneously measuring light reflected from and fluorescence excited in the eye, used as position marker and to track compounds respectively. The measurement concept has been proven by monitoring the diffusion of fluorescein and a pharmaceutical compound for treating open angle glaucoma in vitro in a cuvette and in ex vivo porcine eyes. To obtain a baseline of natural fluorescence we measured the change in corneal and crystalline lens autofluorescence in volunteers over a week. We furthermore present data on 3D ocular autofluorescence. Our results demonstrate the capability to measure the location and concentration of the compound of interest with high axial and temporal resolution of 178 μm and 0.6 s respectively. The current detection limit is 2 nM for fluorescein, and compounds with a quantum yield as low as 0.01 were measured to concentrations below 1 μM. The instrument has many applications in assessing the diffusion of fluorescent compounds through the eye and skin in vitro and in vivo, measuring autofluorescence of ocular tissues and reducing the number of animals needed for research. The instrument has the capability of being used both in the clinical and home care environment opening up the possibility of measuring controlled drug release in a patient friendly manner.
Since their invention by Ashkin optical tweezers have demonstrated their ability and versatility as a non-invasive tool for micromanipulation. One of the most useful additions to the basic optical tweezers system is micro-Raman spectroscopy, which permits highly sensitive analysis of single cells or particles. We report on the development of a dual laser system combining two spatial light modulators to holographically manipulate multiple traps (at
1064nm) whilst undertaking Raman spectroscopy using a 532nm laser. We can thus simultaneously trap multiple particles and record their Raman spectra, without perturbing the trapping system. The dual beam system is built around micro-fluidic channels where crystallisation of calcium carbonate occurs on polymethylmethacrylate (PMMA) beads. The setup is designed to simulate at a microscopic level the reactions that occur on items in a dishwasher, where permanent filming of calcium carbonate on drinking glasses is a problem. Our system allows us to monitor crystal growth on trapped particles in which the Raman spectrum and changes in movement of the bead are recorded. Due to the expected low level of crystallisation on the bead surfaces this allows us to obtain results quickly and with high sensitivity. The long term goal is to study the development of filming on samples in-situ with the microfl.uidic system acting as a model dishwasher.
The ability to image inside the naturally-beating zebrafish heart is becoming increasingly attractive
for developmental and functional biological investigations, but some form of synchronization is
required in order to acquire consistent 3D datasets.
We describe our real-time synchronized selective plane illumination microscope (SPIM), and show
results including the non-invasive acquisition of time-resolved 3D fluorescence images ("4D"
imaging) of the naturally-beating embryonic zebrafish heart. We will also describe the extension of
this technique to synchronized optical intervention.
We report on the incorporation of adaptive optics (AO) into the imaging arm of a selective plane illumination microscope (SPIM). SPIM has recently emerged as an important tool for life science research due to its ability to deliver high-speed, optically sectioned, time-lapse microscope images from deep within in vivo selected samples. SPIM provides a very interesting system for the incorporation of AO as the illumination and imaging paths are decoupled and AO may be useful in both paths. In this paper, we will report the use of AO applied to the imaging path of a SPIM, demonstrating significant improvement in image quality of a live GFP-labeled transgenic zebrafish embryo heart using a modal, wavefront sensorless approach and a heart synchronization method. These experimental results are linked to a computational model showing that significant aberrations are produced by the tube holding the sample in addition to the aberration from the biological sample itself.
We report on the development of a non-invasive instrument based on scanning confocal microscopy for tracking
inherently fluorescent drugs and measuring spatial features in the anterior chamber of the eye. The new instrument
incorporates all features of the initial instrument1 with the addition of fluorescence detection from within the anterior
chamber of the eye. We have measured the diffusion of Fluorescein with high time resolution within a cuvette, an
artificial eye and ex vivo porcine eyes. Results are be presented that demonstrate the capability of the instrument to
accurately measure the concentration and the location of the fluorescent drug over a given period of time along the
optical axis of the eye with an axial resolution of under 200 μm and temporal resolution of < 1s. We show that the
instrument has high sensitivity and can measure concentrations of < 1μM/L of compounds having a quantum yield as
low as 0.01 with high specificity for the compound of interest over competing background signals. The role of the
instrument in assessing the efficiency of any inherently fluorescent ophthalmic drug as well as monitoring other
medication that might produce fluorescent compounds in the eye will be discussed. We furthermore believe that the
instrument might also be capable of monitoring certain bodily processes which have an impact on the compounds present
in the eye.
We report on the development of a widefield microscope that achieves adaptive optics correction through the use of a
wavefront sensor observing an artificial laser guide star induced within the sample. By generating this guide star at
arbitrary positions and depths within the sample we allow the delivery of high-resolution images. This approach delivers
much faster AO correction than image optimization techniques, and allows the use of AO with fluorescent imaging
modalities without generating excessive photo-toxic damage in the sample, or inducing significant photo-bleaching in
the flurophore molecules.
We demonstrate the use of prospective gating from continuously acquired brightfield images of zebrafish embryos to
trigger the acquisition of fluorescence images with the heart at a precisely selected position in its cycle. The laser
exposure of the sample is reduced by an order of magnitude compared to alternative techniques which acquire many
separate fluorescence images for each section before selecting the most appropriate ones to build up a consistent 3D
image stack.
We present results obtained using our SPIM system including 3D reconstructions of the living, beating heart, acquired
using optical gating without the need for any pharmacological or electrophysiological intervention, and discuss possible
wider applications of our technique.
We demonstrate real-time microscope image gating to an arbitrary position in the cycle of the beating heart of a zebrafish embryo. We show how this can be used for high-precision prospective gating of fluorescence image slices of the moving heart. We also present initial results demonstrating the application of this technique to 3-D structural imaging of the beating embryonic heart.
We present results demonstrating for the first time that an optically trapped bead can be used as a local probe to measure
the variation in the viscoelastic properties of the vitreous humor of a rabbit eye. The Brownian motion of the optically
trapped bead was monitored on a fast CCD camera on the millisecond timescale. Analysis of the bead trajectory provides
local information about the viscoelastic properties of the medium surrounding the particle. Previous, bulk, methods for
measuring the viscoelastic properties of the vitreous destroy the sample and allow only a single averaged measurement to
be taken per eye. Whereas, with our approach, we were able to observe local behaviour typical of non-Newtonian and
gel-like materials, along with the homogenous and in-homogeneous nature of different regions of the dissected vitreous
humor. The motivation behind these measurements is to gain a better understanding of the structure of the vitreous
humor in order to design effective drug delivery techniques. In particular, we are interested in methods for delivering
drug to the retina of the eye in order to treat sight threatening diseases such as age related macular degeneration.
The interactions between T cells and antigen-presenting cells (APCs) are crucial in triggering a successful antigenspecific,
adaptive immune response leading to protection against a particular pathogen or disease. At present very little is
known about the magnitudes of the forces involved in these interactions. We present results showing for the first time
that optical tweezers can be used to measure these cell-cell interaction forces. We were able to see a significant
difference in the force distributions taken with and without antigen, as evidenced by a Mann-Whitney U-test. The T cells
of interest were trapped directly and no exogenous beads were added to the sample. Interaction forces between T cells
and APCs in the presence of specific antigen ranged from 0-6.5 pN, whereas, when the specific antigen was absent the
interaction forces ranged from 0-1.5 pN. The accuracy of the system will be discussed in terms of how we tracked the
position of the optically trapped cell and the methods we used to minimise cell roll.
We report on recent developments in the use of adaptive optics (AO) in wide-field microscopy to remove both system
and sample induced aberrations. We describe progress on using both a full AO system and image optimization
techniques (wavefront sensorless AO). In the latter system the determination of the best mirror shape is found via two
routes. In the first an optimization algorithm using a Simplex search pattern is used with an initial random set of mirror
shapes. We then explore the use of specific Zernike terms as our starting basis set. In both cases the final optimization
performance is not affected by the choice of optimization metric. We then describe an open loop AO system in which the
equivalent of a laser guide star is used as the light source for the wavefront sensor.
With the increasing interest in the exploitation of micro-reactors, there is a growing demand for process monitoring and
control methods suitable for application in this environment. At present off-line analysis methods such as
chromatography and mass spectrometry are the dominant tools in the field. Although these methods provide
exceptionally rich chemical information they require removal of samples from the system and the analysis is not
instantaneous. In many microfluidic applications these limitations outweigh their benefits due to the importance of real-time
detection and the desired ability to analyze the fluid in different locations in the micro-reactor non-invasively.
Therefore optical detection methods such as fluorescence and Raman spectroscopy are becoming increasingly popular in
this field, with most attention being drawn to miniature integrated optical sensors. However, integration of sensors into a
micro-reactor can change the flow conditions and make the system difficult to scale out. It is also impossible to move the
integrated sensor along the flow path. These issues make on-chip process analysis a challenging subject that is still at the
early stages of development. This paper discusses opportunities for non-invasive process analysis in micro-reactors
focusing the main attention on Raman spectrometry as a powerful technique, whose potential in this field has not been
widely recognized yet. With a specially developed probe we demonstrate ability to monitor fluid delivery stability and
perform fast real-time analysis of a model esterification reaction. The discussed approach brings unique benefits to
kinetics studies, efficient process optimization and process control.
It has now been widely demonstrated that AO can play a significant role in improving the image quality obtained in
optical microscopes, in particular imaging deeply into biological samples. Although there are differences in the challenge
of overcoming aberrations in optical microscopy and astronomy, for example effects are generally less dynamic in
microscopy; there is a commonality in having to determine the shape to be placed on the adaptive optic element. This
paper briefly reviews the in-depth imaging challenge and then present results on different approaches to determining the
mirror shape and real-time closed loop control of AO in microscopy in both wide-field and beam scanning instruments.
We describe work on producing a selective plane illumination microscope for cardiac imaging in zebra fish embryos.
The system has a novel synchronization system for imaging oscillating structures (e.g. the heart) and will have adaptive
optics for image optimization.
We report on the development of a novel, low cost instrument that is capable of accurately measuring small, short and long term changes in the thickness of the cornea and tear film at high speed. The performance of the instrument was tested by measuring the influence of Allergan's OPTIVETM lubricating eye drops on the thickness of the cornea and tear film. Comparative measurements to quantify the performance were taken using Haag-Streit's LenStar. It was found that the newly developed instrument accurately measured a change in thickness of around 9 μm with an accuracy comparable
to the LenStar, and with a standard deviation of less than 1 micrometer. Since the new instrument was not configured to
resolve the tear film from the cornea, we are not yet able to distinguish the cause of the thickening.
This paper describes our approach to introducing the basic principles of experimental Biophotonics to undergraduates. We have centered on optical microscopy since this is fundamental to most experimental activity associated with Biophotonics whether as a research, diagnostic or therapeutic tool. The major issues associated with imaging include spatial resolution, image enhancement and image interpretation. We have elected to guide students through the principles underlying these concepts by using three linked experimental investigations. The first deals with Fourier Optics and imaging at the fundamental level including the impact of such factors as numerical aperture, illumination wavelength and spatial filtering. The second is an introduction to optical microscopy including the use of digital image capture and basic image manipulation, whilst the third investigates image enhancement techniques such as the use of fluorescent labels and specifically tailored illumination techniques.
For in-vivo imaging applications the use of conventional confocal microscope systems are simply not practical due to
their relatively large size and weight. There is, however, great interest from both the life science research community and
the clinical profession for the development of compact and portable micro-optical instrumentation capable of achieving
minimally invasive, in-vivo imaging of tissue with sub-cellular resolution. In this paper we describe a novel confocal
micro-imaging system incorporating, at its core, a thermally driven, non-resonant two-axis MEMS scanner which serves
as a substitute for the two single-axis galvanometer scanners commonly used in standard confocal imaging systems. In
this paper we describe the non-linearity of such devices and a number of techniques to compensate for this.
We report on the use of adaptive optics in coherent anti-Stokes Raman scattering microscopy (CARS) to improve the
image brightness and quality at increased optical penetration depths in biological material. The principle of the
technique is to shape the incoming wavefront in such a way that it counteracts the aberrations introduced by imperfect
optics and the varying refractive index of the sample. In recent years adaptive optics have been implemented in
multiphoton and confocal microscopy. CARS microscopy is proving to be a powerful tool for non-invasive and label-free
biomedical imaging with vibrational contrast. As the contrast mechanism is based on a 3rd order non-linear optical
process, it is highly susceptible to aberrations, thus CARS signals are commonly lost beyond the depth of ~100 μm in
tissue. We demonstrate the combination of adaptive optics and CARS microscopy for deep-tissue imaging using a
deformable membrane mirror. A random search optimization algorithm using the CARS intensity as the figure of merit
determined the correct mirror-shape in order to correct for the aberrations. We highlight two different methods of
implementation, using a look up table technique and by performing the optimizing in situ. We demonstrate a significant
increase in brightness and image quality in an agarose/polystyrene-bead sample and white chicken muscle, pushing the
penetration depth beyond 200 μm.
We aim to characterize natural caries enamel lesions by fluorescence spectroscopy. Sixty human samples with natural noncavitated caries lesions on smooth surfaces were selected and classified into three groups: dull, shiny, and brown lesions. All the samples were analyzed externally at the natural surface and after hemisectionig internally at the center of the lesion. The lesions were excited with a 405-nm InGaN diode laser and the fluorescence was collected with a single grating spectrometer. Four emission bands (455, 500, 582, and 622 nm) are identified in both sound and carious regions. The area under each emission band is correlated with the total area of the four bands for the sound and carious regions. The detected fluorescence from natural and cut surfaces through the caries lesions is not statistically different for the shiny and dull lesion, but is different [analysis of variance (ANOVA) (p<0.05)] for brown lesion at all emission bands. At the 405-nm excitation wavelength, the area of the fluorescence bands at 455 and 500 nm differ statistically for natural carious lesions and sound tissue.
The Brownian dynamics of an optically trapped water droplet is investigated across the transition from over to
under-damped oscillations. The spectrum of position fluctuations evolves from a Lorentzian shape typical of overdamped
systems (beads in liquid solvents), to a damped harmonic oscillator spectrum showing a resonance peak.
In this later under-damped regime, we excite parametric resonance by periodically modulating the trapping
power at twice the resonant frequency. We also derive from Langevin dynamics an explicit numerical recipe
for the fast computation of the power spectra of a Brownian parametric oscillator. The obtained numerical
predictions are in excellent agreement with the experimental data.
In the 1970s, Jones demonstrated a photon drag by showing that the translation of a window caused a slight displacement
of a transmitted light beam. Similarly he showed that a spinning medium slightly rotated the polarization state. Rather
than translating the medium, the speed of which is limited by mechanical considerations, we translate the image and
measure its lateral delay with respect to a similar image that has not passed through the window. The equivalence, or
lack of it, of the two frames is subtle and great care needs to be taken in determining whether or not similar results are to
be obtained.
In this paper we demonstrate structured illumination microscopy, a cheap and flexible method of obtaining optical
sectioning in wide-field microscopy, and we investigate line-scanning microscopy, both using the same microelement
LED. In our first experiment we demonstrate structured illumination using a custom-designed microelement LED
consisting of 120 individually addressable stripes emitting at 470 nm. An electrical driver was designed to produce a
programmable grid pattern and the device was located in an Olympus BX41 fluorescence microscope in critical
illumination configuration. By using an integrated solid-state alternative to a conventional piezo-actuated grid and
separate illumination source, we improved the speed and accuracy of the system, reducing the artefacts due to the errors
in the grid positions. Additionally, we investigated the use of the same LED device as a scanning source for confocal
line-scanning microscopy. After each line scanning, an image was acquired using a CCD camera and the out-of-focus
light was rejected by a post-processing method.
Sample induced optical aberrations in slices of rat brain tissue have been corrected with a deformable membrane mirror.
The aberration correction required by the DMM was determined using a genetic algorithm with the intensity at a point in
the sample as a fitness value. We show that by optimising on the intensity of a single point in the sample we are able to
improve the axial resolution across the whole field of view of the image at a fixed sample depth. The ratio between the
corrected axial resolution and the diffraction limited resolution is on average 2.7 for a 50 μm thick rat brain tissue sample
and 12 for a 380 μm thick sample across the whole field of view. The uncorrected ratio being 4.1 and 15.5 respectively.
Using a single aberration correction per depth, compared to a point-by-point aberration correction, will significantly
decrease scan times and therefore reduce photobleaching and phototoxic effects enabling more rapid microscopy with
active aberration correction.
We have investigated the application of narrow-band intracavity spectroscopy for high throughput characterization and
detection of reaction products in-situ. An external cavity diode laser (ECDL), tunable from 810-890 nm, is used to
measure varying concentrations of a range of compounds in solution. A linear calibration curve for parts per billion (ppb)
concentrations corresponding to 17 nM has been achieved, levels not detectable with UV/VIS spectrometers. By
replacing the grating with a high reflective mirror, parts per trillion (ppt) concentration detection was also achieved
corresponding to 340 pM. We believe that this technique provides a method for fast and safe measurements of reactions
in real time and has potential applications in the pharmaceutical and chemical industry as well as for integrated bio-diagnostics
lab-on-a-chip devices.
The desire to image with sub micron resolution at ever increasing depths into living samples is providing optical
physicists with the latest in a long line of challenges presented by life science researchers. The advent of confocal, and
subsequently multiphoton microscopy, has opened up exciting new possibilities but simultaneously posed new
challenges. As one images ever more deeply into the sample, the optical properties of the tissue distort the image
significantly lowering the resolution and, in the case of multiphoton imaging in particular, decreasing the fluorescence
yield as the excitation volume rises. The recent use of active optical elements has shown a way forward in restoring high
contrast high resolution images at depth. However, significant issues on the actual shape required on such an element are
as yet unresolved.
We report on two recent advances in this area. The first is the use of a range of optimisation algorithms to restore the
optical point spread function and hence improve the image quality at depth. The second is a radically new approach
incorporating two active elements, a slow spatial light modulator and a fast deformable mirror, to actively lock up the
system. We report on the latest advances in active image compensation where conections at over 5OOmicrons into the
sample have been made using a combination of deformable mirrors and spatial light modulators.
Confocal laser scanning microscopy (CLSM) has rapidly become an essential tool in the life sciences laboratory,
enabling high-resolution and minimally intrusive optical sectioning of fluorescent samples. Most commercially available
CLSM systems employ a gas laser, e.g. a Kr/Ar laser, to provide the excitation radiation. However, such lasers have
several shortcomings, including the maintenance requirements, short lifetimes and high noise levels. To overcome these
limitations, a light source for CLSM that is based on supercontinuum generation in photonic crystal fiber has been
developed. This source provides the necessary wavelength range required to excite the widest possible variety of
fluorophores. A novel method of extracting the desired wavelengths from the supercontinuum source using a digital
micro-mirror device (DMD) is also described.
Since the advent of the optical microscope around 400 years ago there has been an increasing desire by life science researchers to image ever more deeply into samples with high resolution. More recently this desire has lead to the requirement to image three dimensional, living samples with sub-cellular resolution. The application of practical confocal microscopes partially solved this challenge but it was not until the development of multiphoton imaging methods 15 years ago that true in vivo, imaging with high resolution could take place at depth within samples. This paper reviews the basic principles behind multiphoton microscopy and the advances that have been made in the last five years with regard to real time, in depth, imaging. Consideration is given to the best design of multiphoton instruments along with recent research that has been undertaken in the use of active optical elements to enhance in vivo imaging. Some guidelines for the correct selection of the microscopy method for a range of life science challenges are also presented.
Confocal techniques allow the user to achieve optically sectioned images with significantly enhanced axial and improved lateral resolution compared to widefield methods. Unfortunately, as one images more deeply within a sample, sample induced aberrations lead to a significant reduction in image resolution and contrast. Using adaptive optic techniques, we report on the effectiveness of a number of algorithms for removing sample induced aberrations. The viability and efficiency at a number of fitness parameters used in the optimisation routines is also considered.
We present a method for aberration correction in a confocal microscope that successfully combines both a spatial light modulator and a deformable membrane mirror. An active locking technique is used that benefits from the fast update rate of the deformable membrane mirror and the large effective stroke of the spatial light modulator. Concentrating on defocus, we were able to track 'best focus' over a distance of 80 μm with a lock RMS precision of 57 nm. In principle, this technique can be applied to any Zernike mode or aberration that can be accurately reproduced on the deformable membrane mirror.
Evaluation of molar dental restorations on enamel is performed using optical coherence tomography (OCT) with 10 µm resolution. Images of ~50 µm failure gaps in the restorations are demonstrated and the OCT images are compared with x-ray and optical microscopy pictures. The results demonstrate the potential of the technique for clinical evaluation of dental restorations.
We report on the integration of active optical elements in a multiphoton microscope to improve the
resolution and overall image quality when imaging deeply into biological samples. Optical models
were generated of sample systems and these have been compared with the performance of the
complete imaging system. The active elements used were commercially available flexible membrane
mirrors controlled by custom, home written, software. Significant improvements in image quality
have been demonstrated using a range of optimisation routines based on the analysis of the images
produced by the system, rather than with a wavefront sensor. A three-fold increase in the resolution
100μm into the sample was achieved.
We report on the use of a blue laser diode to excite natural fluorescence from a range of dental
samples. Sixty teeth with naturally occurring lesions were examined. The lesions were classified on
visual inspection as falling into three classes as shiny lesions, dull lesions and brown lesions. The
resulting fluorescence was then examined using a fibre optically coupled spectrometer in a series of
wavelength bands. By examining the intensity ratios in the two wavelength bands 480-500nm and
620 to 640nm we were able to classify the lesion types and thus provide a quantifiable measurement
of lesion types. This method has application for detection of dental caries as well as demonstrating
potential application to evaluate lesions which may represent different degrees of caries activity.
Controlled ablation of GaN, sapphire and SiC was investigated using both nanosecond and femtosecond laser pulses. Well-defined patterns of feature size ~10s of microns were successfully machined by fs pulses in all materials. Nanosecond (355nm) machining was primarily successful in machining GaN. Results for the different materials and pulse duration regimes are compared and contrasted.
Preliminary investigations into the potential for automatic spatial-mode optimization in tehrmally distorted soild-sate lasers using deformable mirrors are presented. A 37 element adaptive optic mirror has been used intracavity to control the oscillation mode profile of a diode-laser pumped Nd:YVO4 laser. Spatial mode and output power optimization are demonstrated by closed loop computer control of the deformable mirror using a modified hill-climbing algorithm.
In order to stop or prevent the progression of dental disease, early detection and quantification of decay are crucially important. Dental decay (caries) detection methods have traditionally involved clinical examination by eye, using probes and dental radiography, but up to 60% of lesions are missed. What the dentist requires is a cheap, reliable method of detection of early disease, ideally with information on the depth and rate of growth or healing. Conventional commercial scanning confocal microscopes are unsuitable for use on dental patients. We report on a fibre optic based confocal microscope designed for in vivo examination of caries lesions. The system utilizes a common fibre both as the source and to detect the reflected confocal signal. The initial system has been optimized using dielectric mirrors and the thickness of the stack has been measured with high precision. Dental samples have been examined and the system has been demonstrated to provide information on the depth and mineral loss of a lesion. Fibre optic microscopy (FOCM) demonstrates a practical route to developing an in vivo caries profiler. In this paper, the FOCM and its applications in caries detection are described and the potential of this scheme as a practical dental probe is discussed.
We report on the application of a novel blue laser diode source to confocal microscopy. The source has the potential to be a replacement for argon lasers in a range of fluorescence based imaging systems. It has been demonstrated that with the use of a minimal number of optical components, high quality confocal images can be obtained from laser diodes operating around 406nm. Improvements in image quality through the use of anamorphic prisms to modify the beam profile have been investigated. Living mammalian cells stained with a range of biologically significant compounds have been imaged with high resolution. The stains excited range from fluorescein based compounds to green fluorescent protein. Through the use of the absorption wings a wide range of shorter wavelength fluorophores have been excites, including those more normally excited using UV laser systems. It is expected that this will lead to reduced photo-toxicity within the sample and conventional rather than UV transmitting objective lenses can be used.
A most-important-variables analysis of practical, successful multiphoton excitation fluorescence microscopy is presented. The key strength of multiphoton imaging -- localization of the excitation volume -- helps to decouple the excitation from the emission; the emission no longer needs to be imaged. The presentation starts with maximizing the detected signal and proceeds to the laser source considerations. The main goal is to match the instrumentation to the biological problem. The main aspects covered are... Emission collection: potential signal; Focal plane mismatch: chromatic aberration; Light detected: actual signal; Live cell imaging: preservation of biological function; Deep imaging: preservation of image contrast; Signal production: potential limitations.
Most multiphoton imaging has been undertaken using tuneable femtosecond Ti:Sapphire lasers which are large, expensive and require a level of laser expertise to operate. Although new commercial, computer controlled systems are becoming available they are still complex instruments. We report on the development of a range of novel laser sources for multiphoton microscopy based upon optically pumped semiconductor materials.
We report on the development of practical and user friendly lasers for multiphoton imaging of biological material. The laser developed for the work is a laser diode pumped Cr:LiSAF source modelocked using a saturable Bragg reflector as the passive modelocking element. For this system we routinely obtain 100 fs pulses at a repetition rate 200 MHz with an average output power of 20 mW. The laser has a single operator control and is particularly suitable for use by non-laser specialists. We have used the source developed to image a range of biologically significant samples. The initial work has centered on the imaging of intact human dental tissue. The first two-photon images of dental tissue are reported showing the development of early dental disease from depths up to 500 micrometers into the tooth. These results demonstrate the detection of carious lesions before the more conventional techniques currently used by dental practitioners. Work on other living intact biological tissue is also reported, in particular plants containing a genetically bred fluorescent marker to enable the examination of complete and intact living plant tissue.
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