Photonic tools for advanced sensing and imaging at the nanoscale.

This dissertation reports a novel bio-sensing strategy based on single-mode, electro-active, integrated optical waveguide (SM-EA-IOW) platforms. It also reports the development of a super-resolved far-field optical imaging tool to enable optical, electronic, and spectroelectrochemical investigations at the nanoscale. SM-EA-IOW platforms with its outstanding sensitivity for spectroelectrochemical interrogation was combined with a sandwich bioassay for the development of a novel immunosensing based strategy for label-free detection of infectious pathogens. The strategy begins with the functionalization of the electroactive waveguide surface with a capturing antibody aimed at a specific target analyte. Once the target analyte is bound to the photonic interface, it promotes the binding of a secondary antibody that has been labeled with a redox active reporter. This labeled antibody reporter forms the analytical signal, which is linked uniquely to both the spectral and electrochemical properties of the redox probe designed to specifically recognize a target analyte. Based on this novel detection strategy experimental results in the interrogation of influenza A (H5N1) HA protein have reached an outstanding level of detection in the picomolar range. In addition, the novel label-free SM-EA-IOW bio-sensing strategy was successfully demonstrated for detection of gram-negative bacteria in present authentic clinical eye samples. Such demonstration has also shown the flexibility and ability of the new strategy to probe samples in in the microliter volume range, without any prior processing or pre-enrichment steps. As the groundwork towards the optimal operation of the novel sensing strategy, the effects of the adsorption process and the rate of electron transfer of redox bound species to the electrode surface were thoroughly studied. For each interface of a multilayer immunoassay assembly the surface density, the adsorption kinetic, and the electron-transfer rate of bound species of a redox-active protein were investigated using an optical impedance spectroscopy (OIS) technique based on measurements obtained with the SM-EA-IOW platform. Such methodology and acquired knowledge are crucial for the rational development of novel and advanced immuno-biosensors. Electrochemically modulated fluorescent molecules to be conjugated with relevant antibodies for creating an electroactive probe at the nanoscale was also investigated. Such capability has the potential to enable the development of an arrayed immunosensing technology. Fluorescence emission at the nanoscale suffers from two main restrictions, diffraction limit and photobleaching effects. To address these hinders, a modulated stimulated emission depletion microscope (STED) that is capable of achieving far-field super-resolved images was developed and used to reduce the power of the applied excitation and depletion laser beams diminish photobleaching effects in single-molecule sub-diffraction STED imaging. These two photonic devices provide new approaches for bio-sensing from ensemble range to single molecule detection studies and sensing, where new detection limits can be reached that is expected to establish novel bio-sensing devices with higher sensitivity, specificity and easier ways of sample handling

Isotropic 3D Super-resolution Imaging with a Self-bending Point Spread Function

Airy beams maintain their intensity profiles over a large propagation distance without substantial diffraction and exhibit lateral bending during propagation1-5. This unique property has been exploited for micromanipulation of particles6, generation of plasma channels7 and guidance of plasmonic waves8, but has not been explored for high-resolution optical microscopy. Here, we introduce a self-bending point spread function (SB-PSF) based on Airy beams for three-dimensional (3D) super-resolution fluorescence imaging. We designed a side-lobe-free SB-PSF and implemented a two-channel detection scheme to enable unambiguous 3D localization of fluorescent molecules. The lack of diffraction and the propagation-dependent lateral bending make the SB-PSF well suited for precise 3D localization of molecules over a large imaging depth. Using this method, we obtained super-resolution imaging with isotropic 3D localization precision of 10-15 nm over a 3 μm imaging depth from ∼2000 photons per localization

Development and application of fluorescence lifetime imaging and super-resolution microscopy

This PhD thesis reports the development and application of fluorescence imaging technologies for studying biological processes on spatial scales below the diffraction limit. Two strategies were addressed: firstly fluorescence lifetime imaging (FLIM) to study molecular processes, e.g. using Förster resonance energy transfer (FRET) to read out protein interactions, and secondly direct imaging of nanostructure using super-resolution microscopy (SRM). For quantitative FRET readouts, the development and characterisation of an automated multiwell plate FLIM microscope for high content analysis (HCA) is described. Open source software was developed for the data acquisition and analysis, and approaches to improve the performance of time-gated imaging for FLIM were evaluated including different methods to despeckle the laser illumination and testing of an enhanced detector. This instrument was evaluated using standard fluorescent dye samples and cells expressing fluorescent protein-based FRET constructs. It was applied to an assay of live cells expressing a FRET biosensor and to FRET readouts of aggregation of a membrane receptor (DDR1) in fixed cells. A novel instrument, combining structured illumination microscopy (SIM) with FLIM, was developed to explore the combination of SRM and FLIM-FRET readouts. This enabled the simultaneous mapping of molecular readouts with FLIM and super-resolved imaging. The SIM+FLIM system was applied to image collagen-stimulated DDR1 aggregation in cells, to image DNA structures during the cell cycle and to explore interactions between cell organelles. A novel SRM approach based on a stimulated emission of depletion (STED) microscope incorporating a spatial light modulator (SLM) was developed to provide straightforward robust alignment with collinear excitation/depletion beams, aberration correction, an extended field of view and multiple beam scanning for faster STED image acquisition. The performance of easySLM-STED was evaluated by imaging bead samples, labelled vimentin in Vero cells and the synaptonemal complex in homologs of C. elegans germlines.Open Acces

Development of 3D-STED microscopy and its application to luminescent defects in diamond, nanoparticles and biological samples

The work presented in this thesis follows two main branches. The first aims to develop instrumentation for 3D-STED microscopy and to apply it to the study of bulk diamond, nanoparticles and biological samples. The second aims to evaluate the application of fluorescence imaging and spectroscopy techniques to the study of luminescent defects in diamond. Building on previous work in the Photonics Group at Imperial College London, spatial light modulator (SLM) technology was incorporated into a STED system in a novel configuration to provide a robust and convenient solution for 3D-STED microscopy. This system was applied to the first reported super-resolution imaging of the interaction between two cells in their natural state. The system was further applied to STED imaging of nitrogen vacancy centres in bulk diamond and to a proof of principle experiment for novel plasmon-assisted labels for STED microscopy. The effects of wavefront aberration on STED microscopy were investigated and a predictive correction philosophy was developed based on spherical aberration induced by a refractive index mismatch. The flexibility offered by the SLM technology was taken advantage of to demonstrate recovery of STED imaging quality in glycerol and bulk diamond by active correction of spherical aberration experienced by the depletion point spread function. Confocal intensity imaging, confocal fluorescence lifetime imaging (FLIM) and multispectral fluorescence lifetime measurement were applied to the imaging of fluorescent defects in bulk diamond. It was demonstrated that FLIM can provide information that is complimentary to intensity imaging in diamond and that it is possible to spectrally distinguish defects in diamond while simultaneously measuring their lifetime using multispectral lifetime measurement methods. This thesis also presents the ongoing development of a system for STED of live samples that express green fluorescent protein (GFP).Open Acces

Pushing the physical limits of infrared chemical imaging: intravascular photoacoustic & mid-infrared photothermal

Providing molecular fingerprint information, vibrational spectroscopy is a powerful tool for chemical analysis. In the mid-infrared window, FT-IR spectroscopy and microscopy have been routinely used for sample characterization. In the near-IR window, near-infrared spectroscopy has been widely used for tissue analysis and for the detection of lipids in the arterial walls. Yet, these traditional linear spectroscopies have intrinsic limitations. FT-IR spectroscopy suffers from a poor spatial resolution and strong water absorption for the study of living systems. Near-infrared spectroscopy avoids water absorption, yet it suffers from a poor, millimeter-scale spatial resolution in tissue analysis. My thesis focuses on breaking these limitations through photoacoustic and photothermal detection approaches. The first part of my thesis is on improving the spatial resolution in catheter-based intravascular photoacoustic (IVPA) imaging. By near-infrared excitation of lipids and acoustic detection, IVPA allows depth-resolved identification of lipid-laden atherosclerotic plaque. Thus far, most IVPA endoscopes use multimode fibers, which do not allow tight focusing of photons. Recent experiments on pulse propagation in multimode graded-index fibers have shown a nonlinear improvement in beam quality. Here, we harness this nonlinear phenomenon for the fiber-delivery of nanosecond laser pulses. We built a photoacoustic catheter 1.4 mm outer diameter, offering a lateral resolution as fine as 30 μm within a depth range of 2.5 mm. Such resolution is one order of magnitude better than current multi-mode fiber-based intravascular photoacoustic catheters. At the same time, the delivered pulse energy can reach as high as 20 μJ, which is two orders of magnitude higher than that of an optical resolution photoacoustic endoscope built with single-mode fiber. These improvements are expected to promote the biomedical application of photoacoustic endoscopes which require both high resolution and high pulse energy. Based on the technical advances, my thesis work further demonstrated longitudinal imaging of the same plaque in the same living animal. Recently developed mid-infrared photothermal (MIP) microscopy overcomes the limitations in FT-IR microscopy by probing the IR absorption-induced photothermal effect using visible light. MIP microscopy yields sub-micrometer spatial resolution with high spectral fidelity and much-reduced water background. The second part of my thesis work pushes the physical limits of MIP microscopy in aspects of detection sensitivity and imaging speed using two approaches. First, taking advantage of the interference scattering effect, the scattering signal from the sample can be greatly enhanced. Together with the relatively large infrared absorption coefficient, the sensitivity of the infrared spectrum is greatly improved, and single virus detection is achieved. Second, by using fluorescence as a thermo-sensitive probe, the temperature raise by infrared absorption can be retrieved in a more efficient way and much higher imaging speed and sensitivity are thus accomplished

Fiber Laser Based Nonlinear Spectroscopy

To date, nonlinear spectroscopy has been considered an expensive technique and confined mostly to experimental laboratory settings. Over recent years, optical-fiber lasers that are highly reliable, simple to operate and relatively inexpensive have become commercially available, removing one of the major obstacles to widespread utilization of nonlinear optical measurement in biochemistry. However, fiber lasers generally offer relatively low output power compared to lasers traditionally used for nonlinear spectroscopy, and much more careful design is necessary to meet the excitation power thresholds for nonlinear signal generation. On the other hand, reducing the excitation intensity provides a much more suitable level of user-safety, minimizes damage to biological samples and reduces interference with intrinsic chemical processes. Compared to traditional spectroscopy systems, the complexity of nonlinear spectroscopy and imaging instruments must be drastically reduced for them to become practical. A nonlinear spectroscopy tool based on a single fiber laser, with electrically controlled wavelength-tuning and spectral resolution enhanced by a pulse shaping technique, will efficiently produce optical excitation that allows quantitative measurement of important nonlinear optical properties of materials. The work represented here encompasses the theory and design of a nonlinear spectroscopy and imaging system of the simplest architecture possible, while solving the difficult underlying design challenges. With this goal, the following report introduces the theories of nonlinear optical propagation relevant to the design of a wavelength tunable system for nonlinear spectroscopy applications, specifically Coherent Anti-Stokes Spectroscopy (CARS) and Förster Resonance Energy Transfer (FRET). It includes a detailed study of nonlinear propagation of optical solitons using various analysis techniques. A solution of the generalized nonlinear Schrödinger equation using the split-step Fourier method is demonstrated and investigation of optical soliton propagation in fibers is carried out. Other numerical methods, such as the finite difference time domain approach and spectral-split step Fourier methods are also described and compared. Numerical results are contrasted with various measurements of wavelength shifted solitons. Both CARS and FRET test-bed designs and experiments are presented, representing two valuable biochemical measurement applications. Two-photon excitation experiments with a simplified calibration process for quantitative FRET measurement were conducted on calmodulin proteins modified with fluorescent dyes, as well as modified enhanced green fluorescent protein. The resulting new FRET efficiency measurements showed agreement with those of alternative techniques which are slower and can involve destruction of the sample. In the second major application of the nonlinear spectroscopy system, CARS measurement with enhanced spectral resolution was conducted on cyclohexane as well as on samples of mouse brain tissue containing lipids with Raman resonances. The measurements of cyclohexane verified the ability of the system to precisely determine its Raman resonances, thus providing a benchmark within a similar spectral range for biological materials which have weaker Raman signal responses. The improvement of spectral resolution (resonance frequency selectivity), was also demonstrated by measuring the closely-spaced resonances of cyclohexane. Finally, CARS measurements were also made on samples of mouse brain tissue which has a lipids-based Raman signature. The CARS spectrum of the lipid resonances matched well with other cited studies. The imaging of mouse brain tissue with Raman resonance contrast was also partially achieved, but it was hindered by low signal to noise ratio and limitations of the control hardware that led to some dropout of the CARS signal due to power coupling fluctuations. Nevertheless, these difficulties can be straightforwardly addressed by refinement of the wavelength tuning electronics. In conclusion, it is hoped that these efforts will lead to greater accessibility and use of CARS, FRET and other nonlinear spectral measurement instruments, in line with the promising advances in optics and laser technology