Laser Scanning Microscopy with SPAD Array Detector: Towards a New Class of Fluorescence Microscopy Techniques

Laser scanning microscopy is one of the most common architectures in fluorescence microscopy. In a nutshell: the objective lens focuses the laser beam(s) and generates an effective excitation spot which is scanned on the sample; for each pixel, the fluorescent image is projected into a single-element detector, which \u2013 typically \u2013 spatially and temporally integrates the fluorescent light along its sensitive area and the pixel dwell-time, thus providing a single-intensity value per pixel. Notably, the integration performed by the single-element detector hinders any additional information potentially encoded in the dynamic and image of the fluorescent spot. To address this limitation, we recently upgraded the detection unit of a laser scanning microscope, replacing the single-element detector with a novel SPAD (single photon avalanche diode) array detector. We have shown at first that the additional spatial information allows to overcome the trade-off between resolution and signal-to-noise ratio proper of confocal microscopy: indeed, this architecture represents the natural implementation of image scanning microscopy (ISM). We then exploited the single-photon-timing ability of the SPAD array detector elements to combine ISM with fluorescence lifetime imaging: the results show higher resolution and better lifetime accuracy with respect to the confocal counterpart. Moreover, we explored the combination of our ISM platform with stimulated emission depletion (STED) microscopy, to mitigate the non-negligible chance of photo-damaging a sample. Lastly, we showed how the SPAD array-based microscope can be used in the context of single-molecule/particle tracking (SMT or SPT) and spectroscopy. Indeed, we implemented a real-time, feedback based SMT architecture which can potentially correlate the dynamics of a bio-molecule with its structural changes and micro-environment, taking advantage of the time-resolved spectroscopy ability of the novel detector. We believe that this novel laser scanning microscopy architecture has everything in its favour to substitute current single-element detector approaches; it will enable for a new class of fluorescence microscopy techniques capable of investigating complex living biological samples with unprecedented spatial and temporal characteristics and augmented information content

Development of a 1D phased ultrasonic array for intravascular sonoporation

Error on title page – year of award is 2021.Sonoporation represents a promising approach to increase targeted drug delivery efficiency by facilitating transport of therapeutic agents to the target tissue with the use of ultrasound. However, most of the current research in sonoporation is performed with external ultrasonic transducers, which hinders the applicability of the therapeutic procedure for treatment of conditions situated deeper into the patient’s body, such as liver or intestinal tumours. This Thesis presents the development process of a miniature-sized 1-3 connectivity piezocomposite 1D phased array for intracorporeal sonoporation. The device was to be incorporated into a capsule or catheter and hence the primary design constraint was the reduced size of the piezoelectric element, which was limited to 2.5 mm in width and 12 mm in length. To meet the needs of the intended application, resonance frequencies of 1.5 MHz and 3.0 MHz were considered. A simulation framework was developed for optimization of the miniature array in relation to the peak negative pressure attained at the focus to mitigate the low power output associated with the limited device dimensions. This was implemented through a multiparametric sweep of the 1-3 piezocomposite geometry-related parameters. Devices made with PZT-5H and PMN-29%PT were evaluated. The optimization algorithm was used to determine specifications for phased array designs based on the two materials and the two resonance frequencies. The 1.5 MHz devices comprised 24 elements and the 3.0 MHz ones had 32 elements. The piezocomposites were manufactured using the dice and fill technique and electroded using a novel method of electrode deposition employing spin coating of Ag ink. Subsequently, the prototype devices were driven with a commercial array controller and characterized with a calibrated needle hydrophone in a scanning tank. Two simulation profiles based on finite element analysis and time extrapolation were developed to model the acoustic beams from the arrays, which were compared and calibrated with experimental data for focal distances between 5 mm and 10 mm and beam steering angles from 0° to 40°. The results showed that modelling could be employed reliably for therapeutic planning. Both the 1.5 MHz and the 3.0 MHz, PZT-5H arrays were tested in vitro and shown to induce and control sonoporation of a human epithelial colorectal adenocarcinoma cell layer. Finally, a 24 element, 1.5 MHz, PZT-5H array was implemented in a 40 mm long by 11 mm diameter tethered, biocompatible capsule intended for in vivo operation. The device was characterized in the scanning tank for steering angles in the range 0° to 56° and focal distances between 4.0 mm and 5.7 mm, and the measured beam profiles were correlated with the simulation framework. The capsule will be tested in future ex-vivo and in-vivo experiments on insulin absorption through porcine small bowel by means of sonoporation.Sonoporation represents a promising approach to increase targeted drug delivery efficiency by facilitating transport of therapeutic agents to the target tissue with the use of ultrasound. However, most of the current research in sonoporation is performed with external ultrasonic transducers, which hinders the applicability of the therapeutic procedure for treatment of conditions situated deeper into the patient’s body, such as liver or intestinal tumours. This Thesis presents the development process of a miniature-sized 1-3 connectivity piezocomposite 1D phased array for intracorporeal sonoporation. The device was to be incorporated into a capsule or catheter and hence the primary design constraint was the reduced size of the piezoelectric element, which was limited to 2.5 mm in width and 12 mm in length. To meet the needs of the intended application, resonance frequencies of 1.5 MHz and 3.0 MHz were considered. A simulation framework was developed for optimization of the miniature array in relation to the peak negative pressure attained at the focus to mitigate the low power output associated with the limited device dimensions. This was implemented through a multiparametric sweep of the 1-3 piezocomposite geometry-related parameters. Devices made with PZT-5H and PMN-29%PT were evaluated. The optimization algorithm was used to determine specifications for phased array designs based on the two materials and the two resonance frequencies. The 1.5 MHz devices comprised 24 elements and the 3.0 MHz ones had 32 elements. The piezocomposites were manufactured using the dice and fill technique and electroded using a novel method of electrode deposition employing spin coating of Ag ink. Subsequently, the prototype devices were driven with a commercial array controller and characterized with a calibrated needle hydrophone in a scanning tank. Two simulation profiles based on finite element analysis and time extrapolation were developed to model the acoustic beams from the arrays, which were compared and calibrated with experimental data for focal distances between 5 mm and 10 mm and beam steering angles from 0° to 40°. The results showed that modelling could be employed reliably for therapeutic planning. Both the 1.5 MHz and the 3.0 MHz, PZT-5H arrays were tested in vitro and shown to induce and control sonoporation of a human epithelial colorectal adenocarcinoma cell layer. Finally, a 24 element, 1.5 MHz, PZT-5H array was implemented in a 40 mm long by 11 mm diameter tethered, biocompatible capsule intended for in vivo operation. The device was characterized in the scanning tank for steering angles in the range 0° to 56° and focal distances between 4.0 mm and 5.7 mm, and the measured beam profiles were correlated with the simulation framework. The capsule will be tested in future ex-vivo and in-vivo experiments on insulin absorption through porcine small bowel by means of sonoporation

Advances in the Direct Spectral Estimation of Acoustic Sources Using Continuous-Scan Phased Arrays

The present study is related to the field of imaging of aeroacoustic noise sources. Traditional techniques include the use of phased microphone arrays and acoustic beamforming of the signals signals using algorithms such as the Delay-And-Sum (DAS). Over the last years, there has been an increasing interest in methods in which some of the sensors traverse in prescribed paths and motion. Some of the challenges of this approach include the treatment of the non-stationarity of the signal due to the motion of the microphone(s).An objective of this work is to review the methodology presented by D. Papamoschou, P. Shah and myself in the AIAA Journal "Inverse Acoustic Methodology for Continuous-Scan Phased Arrays" since it provides the building grounds for the thesis. The methodology accounts for the direct estimation of the spatio-spectral distribution of an acoustic source from microphone measurements that include fixed and continuously scanning sensors. The non-stationarity of the signal is addressed by means of the Wigner-Ville spectrum. Suppression of the non-stationary effects involves the division of the signal into blocks and the application of a frequency-dependent window within each block. The direct estimation approach involves the inversion of an integral that relates the modeled pressure field, the measured pressure field and the response of the array. A Bayesian-estimation that allows for efficient inversion of the integrals and performs similarly to the conjugate gradient method is reviewed.The coherence-based noise source distribution is studied in this work and the influence of the signal segmentation on its spatial resolution is analyzed. This thesis provides specific guidelines related to the signal processing. The signal is divided into blocks meeting a desired mathematical condition. A minimum and maximum size for the resulting blocks is proposed in this work, as well as a minimum and maximum block overlap. A safe region for the signal segmentation is presented as well.This work presents a methodology to synchronize the signals from the microphones (scanning or not) with the position of the scanning sensor. It also shows the methods to check the accuracy of the position scanning sensor.The methodology is applied to acoustic fields emitted by impinging jets approximating a point source and an overexpanded supersonic jet. Noise source maps that included the scanning sensor and a dense block distribution have increased spatial resolution and reduced sidelobes. The ability of the continuous scan paradigm to provide high-definition noise source maps with a lower sensor count is confirmed in this work as well. The effect of the proposed signal segmentation on sparse arrays is discussed

Characterization and prototyping of the rotating modulator hard x-ray/gamma-ray telescope

A hard x-ray/gamma-ray telescope with high sensitivity and wide field of view would be capable of performing an all-sky census of black holes over a wide range of obscuration and accretion rates. As an example, NASA\u27s Black Hole Finder Probe mission was designed to provide a 5-sigma flux sensitivity in a 1-year observation of ~0.02 mCrab in the 10 - 150 keV energy range and 0.5 mCrab in the 150 - 600 keV energy range with 3 - 5 minutes of arc angular resolution. These are significantly higher sensitivity and resolution goals than those of current instruments. The design focus on sensitivity would make the instrument equally suitable for national security applications in the detection of weak shielded illicit radioactive materials at large distances (100 m - 1 km). X-ray and gamma-ray imaging designs for astrophysics and security applications typically utilize a coded aperture imaging technique. The spatial resolution necessary, however, coupled with the specification of high sensitivity, requires a large number of readout channels (resulting in high cost and complexity) and limits the use of this technique to relatively low energies. As an alternative approach, an investigation is made here of the rotating modulator (RM), which uses primarily temporal modulation to record an object scene. The RM consists of a mask of opaque slats that rotates above an array of detectors. Time histories of counts recorded by each detector are used to reconstruct the object scene distribution. Since a full study of RM characterization and capabilities has not been performed prior to this work, a comprehensive analytic system response is presented, which accounts for realistic modulation geometries. The RM imaging characteristics and sensitivity are detailed, including a comparison to more common hard x-ray imaging modalities. A novel image reconstruction algorithm is developed to provide noise-compensation, super-resolution, and high fidelity. A laboratory prototype RM and its measurement results are presented. As a pathfinder mission to an eventual astrophysics campaign, a one-day high-altitude balloon-borne RM is also described, including expected performance and imaging results. Finally, RM designs suitable for next-generation astrophysics and security applications are presented, and improvements to the RM technique are suggested

Laser-generated, plane-wave, broadband ultrasound sources for metrology

The accurate quantification of ultrasound fields generated by diagnostic and therapeutic transducers is critical for patient safety. This requires hydrophones calibrated to a traceable national measurement standard over the full range of frequencies used. At present, the upper calibration frequency range available to the user community is limited to a frequency of 60 MHz. However, there is often content at frequencies higher than this, e.g., through nonlinear propagation of high-amplitude pulses or tone-bursts for therapeutic applications, and the increasing use of higher frequencies in diagnostic imaging. To reduce the uncertainties and extend the calibrations to higher frequencies, a source of high-pressure, plane-wave and broadband ultrasound fields is required. This is not possible with current piezoelectric transducer technology, therefore laser-generated ultrasound is investigated as an alternative. This consists of an ultrasound wave generated by the pulsed laser excitation of a thin, planar, layer of light absorbing carbon-polymer nanocomposite materials. The work described in this thesis can be divided into three parts. The first part consisted of the fabrication of various nanocomposites in order to study the effect of different polymer types, composite thickness, laser fluence, and concentration of carbon nanotubes, on the ultrasound generated, as well as their stability. This included an investigation into the nonlinear propagation of MPa range laser-generated ultrasound, and the effect of the bandlimited hydrophone response, using a numerical wave solver (k-Wave). In the second part, the effects on the signal of acoustically reflective and matched backings (the substrates onto which the nanocomposite was coated) were studied. It was found experimentally that the backing material can significantly affect the pressure amplitude when the duration of the laser pulse is longer than the acoustic transit time across the thin nanocomposite layer. An analytical model was developed to describe how the signal generated depends on the backing material, absorbing layer thickness, and laser pulse duration. The model agreed well with measurements performed with a variable pulse duration fibre-laser. Finally, in the third part, a laser-generated, plane-wave, broadband ultrasound source device superficially resembling a standard piezoelectric piston source was designed, fabricated, and tested. The source produced quasi-unipolar pressure-pulse of 9 MPa peak-positive pressure with a bandwidth of 100 MHz, and the ultrasound beam is sufficiently planar to reduce uncertainties due to diffraction to negligible levels for hydrophones up to 0.6 mm in diameter

Non-negative matrix decomposition approaches to frequency domain analysis of music audio signals

On étudie l’application des algorithmes de décomposition matricielles tel que la Factorisation Matricielle Non-négative (FMN), aux représentations fréquentielles de signaux audio musicaux. Ces algorithmes, dirigés par une fonction d’erreur de reconstruction, apprennent un ensemble de fonctions de base et un ensemble de coef- ficients correspondants qui approximent le signal d’entrée. On compare l’utilisation de trois fonctions d’erreur de reconstruction quand la FMN est appliquée à des gammes monophoniques et harmonisées: moindre carré, divergence Kullback-Leibler, et une mesure de divergence dépendente de la phase, introduite récemment. Des nouvelles méthodes pour interpréter les décompositions résultantes sont présentées et sont comparées aux méthodes utilisées précédemment qui nécessitent des connaissances du domaine acoustique. Finalement, on analyse la capacité de généralisation des fonctions de bases apprises par rapport à trois paramètres musicaux: l’amplitude, la durée et le type d’instrument. Pour ce faire, on introduit deux algorithmes d’étiquetage des fonctions de bases qui performent mieux que l’approche précédente dans la majorité de nos tests, la tâche d’instrument avec audio monophonique étant la seule exception importante.We study the application of unsupervised matrix decomposition algorithms such as Non-negative Matrix Factorization (NMF) to frequency domain representations of music audio signals. These algorithms, driven by a given reconstruction error function, learn a set of basis functions and a set of corresponding coefficients that approximate the input signal. We compare the use of three reconstruction error functions when NMF is applied to monophonic and harmonized musical scales: least squares, Kullback-Leibler divergence, and a recently introduced “phase-aware” divergence measure. Novel supervised methods for interpreting the resulting decompositions are presented and compared to previously used methods that rely on domain knowledge. Finally, the ability of the learned basis functions to generalize across musical parameter values including note amplitude, note duration and instrument type, are analyzed. To do so, we introduce two basis function labeling algorithms that outperform the previous labeling approach in the majority of our tests, instrument type with monophonic audio being the only notable exception