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published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

A major limitation in tissue engineering is the lack of nondestructive methods to assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a scanning fiber imaging method capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque vascular scaffold, contained in a bioreactor. This imaging modality is based on local excitation of fluorescent cells, acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells, stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled endothelial cells on the luminal surface through a ~500 µm thick tubular scaffold at cell-level resolutions and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution of the order of 20-30 µm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable nondestructive monitoring of endothelial cells seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future. / Ph. D.

Desorption electrospray ionization (DESI) is an ambient ionization technique used for mass spectrometric imaging of biological samples. When coupled with isotopic ratio measurements of deuterium-labeled tissues, DESI provides a means of measuring metabolic rates on a spatially resolved basis. In vivo metabolic rates are desired to better understand diseases such as Alzheimer's, Parkinson's, Huntington's, and various forms of cancer that negatively impact metabolic rates within different organs of the human body. Although DESI has been used to image lipids and metabolites of a variety of tissues and other imaging techniques, such as NIMS, have been used to study kinetic turnover rates, DESI has not yet been used to study in vivo metabolic rates using deuterium labeled tissue. This thesis describes how we optimized our DESI source for imaging of biological tissue, how we developed a MATLAB graphical user interface (GUI) to process and interpret the large mass spectral data files, how we conducted our initial mouse brain study for proof-of-concept, and how we plan to implement our DESI imaging in a study with mouse models of Alzheimer's disease. Our initial mouse brain study involved labeling mice with deuterium enriched water, preparing tissue slices for DESI analysis, imaging the tissue slices using DESI coupled with a Bruker mass spectrometer, analyzing the mass spectral data using our custom-designed image_inspector program, confirming identification of lipids using MS/MS, and creating incorporation curves to measure in vivo metabolic rates.

Cancer is a major health problem in the world today. Almost all cancers have a significantly better chance for therapy and recovery if detected at their early stage. The capability to perform disease diagnosis at an early stage requires high-resolution imaging that can visualise the physiological and morphological changes at a cellular level. However, resolving powers of current medical imaging systems are limited to sub-millimeter sizes. Furthermore, the majority of cancers are associated with morphological and functional alterations of cells in epithelial tissue, currently assessed by invasive and time-consuming biopsy. Optical imaging enables visualisations of tissue microstructures at the level of histology in non-invasive means. Optical imaging is suitable for detecting neoplastic changes with sub-cellular resolution in vivo without the need for biopsy. Nonlinear optical microscopy based on multi-photon absorption and higher harmonic generation has provided spectacular sights into visualisation of cellular events within live tissue due to advantages of an inherent sectioning ability, the relatively deep optical penetration, and the direct visualisation of intrinsic indicators. Two-photon excited uorescence (TPEF) from intrinsic cell components and second harmonic from asymmetric supermolecular structures can provide complementary information regarding functionalities and morphologies in tissue environments, thus enabling premalignant diagnosis by detecting the very earliest changes in cellular structures. During the past sixteen years, nonlinear optical microscopy has evolved from a photonic novelty to a well-established laboratory tool. At present, in vivo imaging and long-term bedside studies by use of nonlinear optical microscopy have been limited due to the fact that the lack of the compact nonlinear optical instrument/imaging technique forces the performance of nonlinear optical microscopy with bulk optics on the bench top. Rapid developments of fibre-optics components in terms of growing functionalities and decreasing sizes provide enormous opportunities for innovation in nonlinear optical microscopy. Fibre-based nonlinear optical endoscopy will be the soul instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible with a conventional optical microscope. Lots of efforts have been made for development of miniaturised nonlinear optical microscopy. However, there are major challenges remaining to create a nonlinear optical endoscope applicable within internal cavities of a body. First, an excitation laser beam with an ultrashort pulse width should be delivered eciently to a remote place where ecient collection of faint nonlinear optical signals from biological samples is required. Second, laser-scanning mechanisms adopted in such a miniaturised instrumentation should permit size reduction to a millimeter scale and enable fast scanning rates for monitoring biological processes. Finally, the design of a nonlinear optical endoscope based on micro-optics must maintain great exibility and compact size to be incorporated into endoscopes to image internal organs. Although there are obvious diculties, development of fibre-optic nonlinear optical microscopy/endoscopy would be indispensible to innovate conventional nonlinear optical microscopy, and therefore make a significant impact on medical diagnosis. The work conducted in this thesis demonstrates the new capability of nonlinear optical endoscopy based on a single-mode fibre (SMF) coupler or a double-clad photonic crystal fibre (PCF), a microelectromechanical system (MEMS) mirror, and a gradientindex (GRIN) lens. The feasibility of all-fibre nonlinear optical endoscopy is also demonstrated by the further integration of a double-clad PCF coupler. The thesis concentrates on the following key areas in order to exploit and understand the new imaging modality. It has been known from the previous studies that an SMF coupler is suitable for twoii photon excitation by transmitting near infrared illumination and collecting uorescence at visible wavelength as well. Although second harmonic generation (SHG) wavelength is farther away from the designed wavelength of the fibre coupler than that of normal TPEF, it is demonstrated in this thesis that both SHG and TPEF signals can be collected simultaneously and eciently through an SMF coupler with axial resolution of 1.8 um and 2.1 um, respectively. The fibre coupler shows a unique feature of linear polarisation preservation along the birefringent axis over the near infrared and the visible wavelength regions. Therefore, SHG polarisation anisotropy can be potentially extracted for probing the orientation of structural proteins in tissue. Furthermore, this thesis shows the characterisation of nonlinear optical microscopy based on the separation distance of an SMF coupler and a GRIN lens. Consequently, the collection of nonlinear signals has been optimised after the investigation of the intrinsic trade-off between signal level and axial resolution. These phenomena have been theoretically explored in this thesis through formalisation and numerical analysis of the three-dimensional (3D) coherent transfer function for a SHG microscope based on an SMF coupler. It has been discovered that a fibreoptic SHG microscope exhibits the same spatial frequency passband as that of a fibreoptic reection-mode non-uorescence microscope. When the numerical aperture of the fibre is much larger than the convergent angle of the illumination on the fibre aperture, the performance of fibre-optic SHG microscopy behaves as confocal SHG microscopy. Furthermore, it has been shown in both analysis and experiments that axial resolution in fibre-optic SHG microscopy is dependent on the normalised fibre spot size parameters. For a given illumination wavelength, axial resolution has an improvement of approximately 7% compared with TPEF microscopy using an SMF coupler. Although an SMF enables the delivery of a high quality laser beam and an enhanced sectioning capability, the low numerical aperture and the finite core size of an SMF give rise to a restricted sensitivity of a nonlinear optical microscope system. The key innovation demonstrated in this thesis is a significant signal enhancement of a nonlinear optical endoscope by use of a double-clad PCF. This thesis has characterised properties of our custom-designed double-clad PCF in order to construct a 3D nonlinear optical microscope. It has been shown that both the TPEF and SHG signal levels in a PCF-based system that has an optical sectioning property for 3D imaging can be significantly improved by two orders of magnitude in comparison with those in an SMF-based microscope. Furthermore, in contrast with the system using an SMF, simultaneous optimisations of axial resolution and signal level can be obtained by use of double-clad PCFs. More importantly, using a MEMS mirror as the scanning unit and a GRIN lens to produce a fast scanning focal spot, the concept of nonlinear optical endoscopy based on a double-clad PCF, a MEMS mirror and a GRIN lens has been experimentally demonstrated. The ability of the nonlinear optical endoscope to perform high-resolution 3D imaging in deep tissue has also been shown. A novel three-port double-clad PCF coupler has been developed in this thesis to achieve self-alignment and further replace bulk optics for an all-fibre endoscopic system. The double-clad PCF coupler exhibits the property of splitting the laser power as well as the separation of a near infrared single-mode beam from a visible multimode beam, showing advantages for compact nonlinear optical microscopy that cannot be achieved from an SMF coupler. A compact nonlinear optical microscope based on the doubleclad PCF coupler has been constructed in conjunction with a GRIN lens, demonstrating high-resolution 3D TPEF and SHG images with the axial resolution of approximately 10 m. Such a PCF coupler can be useful not only for a fibre-optic nonlinear optical probe but also for double-clad fibre lasers and amplifiers. The work presented in this thesis has led to the possibility of a new imaging device to complement current non-invasive imaging techniques and optical biopsy for cancer detection if an ultrashort-pulsed fibre laser is integrated and the commercialisation of the system is achieved. This technology will enable in vivo visualisations of functional and morphological changes of tissue at the microscopic level rather than direct observations with a traditional instrument at the macroscopic level. One can anticipate the progress in bre-optic nonlinear optical imaging that will propel imaging applications that require both miniaturisation and great functionality.

Conventional charged-particle imaging techniques--such as autoradiography--provide only two-dimensional (2D) images of thin tissue slices. To get volumetric information, images of multiple thin slices are stacked. This process is time consuming and prone to distortions, as registration of 2D images is required. We propose a direct three-dimensional (3D) autoradiography technique, which we call charged-particle emission tomography (CPET). This 3D imaging technique enables imaging of thick sections, thus increasing laboratory throughput and eliminating distortions due to registration. In CPET, molecules or cells of interest are labeled so that they emit charged particles without significant alteration of their biological function. Therefore, by imaging the source of the charged particles, one can gain information about the distribution of the molecules or cells of interest. Two special case of CPET include beta emission tomography (BET) and alpha emission tomography (𝛼ET), where the charged particles employed are fast electrons and alpha particles, respectively. A crucial component of CPET is the charged-particle detector. Conventional charged-particle detectors are sensitive only to the 2-D positions of the detected particles. We propose a new detector concept, which we call particle-processing detector (PPD). A PPD measures attributes of each detected particle, including location, direction of propagation, and/or the energy deposited in the detector. Reconstruction algorithms for CPET are developed, and reconstruction results from simulated data are presented for both BET and 𝛼ET. The results show that, in addition to position, direction and energy provide valuable information for 3D reconstruction of CPET. Several designs of particle-processing detectors are described. Experimental results for one detector are discussed. With appropriate detector design and careful data analysis, it is possible to measure direction and energy, as well as position of each detected particle. The null functions of CPET with PPDs that measure different combinations of attributes are calculated through singular-value decomposition. In general, the more particle attributes are measured from each detection event, the smaller the null space of CPET is. In other words, the higher dimension the data space is, the more information about an object can be recovered from CPET.

Microscopic imaging in the biomedical sciences allows for detailed study of the structure and function of normal and abnormal (i.e., diseased) states of cells and tissues. The expression patterns of proteins and/or physiological parameters within these specimens can be related to disease progression and prognosis, and are often heterogeneously spread throughout the entire specimen. With conventional microscopy, a large number of individual image ‘tiles’ must be captured and subsequently combined into a mosaic of the entire specimen. This has the potential to introduce artefacts at the image seams, as well as introducing non-uniform illumination of the entire specimen. A further limitation often encountered in biomedical fluorescence microscopy is the high background due to the autofluorescence (AF) of endogenous compounds within cells and tissues. Often, AF can prevent the detection and/or accurate quantification in fluorescently- labelled tissues and, in general, can reduce the reliability of results obtained from such specimens. AF spectra are relatively broad and so can be present across a large number of image spectral channels. The intensity of AF also increases as the excitation wavelength is decreased, causing increasing amounts of autofluorescence when exciting in the blue and near-UV range of the spectrum (400 - 500 nm). This thesis reports the development of hyperspectral, fluorescence and brightfield imaging of entire, paraffin-embedded, formalin-fixed (PEFF) tissue slides using a prototype confocal scanner with a large field of view (FOV). This technology addresses the challenges of imaging large tissue sections through the use of a telecentric f-theta laser scan lens thus allowing an entire microscope slide (22x70 mm) to be imaged in a single scan at resolution equivalent to a 10x microscope objective. The development and optimization of brightfield and single-channel fluorescence imaging modes are discussed in the first half of this thesis, while the second half and appendices concentrate on the spectral properties of the system and removal of AF from PEFF tissue sections. The hyperspectral imaging mode designed for this system allows the fluorescence emission spectrum of each image pixel to be sampled at 6.7 nm/channel over a spectral range of 500-700 nm. This results in the ability to separate distinct fluorescence signatures from each other, and enables quantification even in situations where the AF completely masks the signal from the applied labels.

Microscopic imaging in the biomedical sciences allows for detailed study of the structure and function of normal and abnormal (i.e., diseased) states of cells and tissues. The expression patterns of proteins and/or physiological parameters within these specimens can be related to disease progression and prognosis, and are often heterogeneously spread throughout the entire specimen. With conventional microscopy, a large number of individual image ‘tiles’ must be captured and subsequently combined into a mosaic of the entire specimen. This has the potential to introduce artefacts at the image seams, as well as introducing non-uniform illumination of the entire specimen. A further limitation often encountered in biomedical fluorescence microscopy is the high background due to the autofluorescence (AF) of endogenous compounds within cells and tissues. Often, AF can prevent the detection and/or accurate quantification in fluorescently- labelled tissues and, in general, can reduce the reliability of results obtained from such specimens. AF spectra are relatively broad and so can be present across a large number of image spectral channels. The intensity of AF also increases as the excitation wavelength is decreased, causing increasing amounts of autofluorescence when exciting in the blue and near-UV range of the spectrum (400 - 500 nm). This thesis reports the development of hyperspectral, fluorescence and brightfield imaging of entire, paraffin-embedded, formalin-fixed (PEFF) tissue slides using a prototype confocal scanner with a large field of view (FOV). This technology addresses the challenges of imaging large tissue sections through the use of a telecentric f-theta laser scan lens thus allowing an entire microscope slide (22x70 mm) to be imaged in a single scan at resolution equivalent to a 10x microscope objective. The development and optimization of brightfield and single-channel fluorescence imaging modes are discussed in the first half of this thesis, while the second half and appendices concentrate on the spectral properties of the system and removal of AF from PEFF tissue sections. The hyperspectral imaging mode designed for this system allows the fluorescence emission spectrum of each image pixel to be sampled at 6.7 nm/channel over a spectral range of 500-700 nm. This results in the ability to separate distinct fluorescence signatures from each other, and enables quantification even in situations where the AF completely masks the signal from the applied labels.

Breast cancer is the most common form of cancer among women, this type of cancer is diagnosed in around 9000 women every year in Sweden. The most common studies to find breast cancer is through mammography where the breast tissue is compressed and exposed by radiation. Not only does the technique expose the breast tissue for radiation, but it can also be very uncomfortable. There is research on a new kind of scanning where use of microwaves reduces the uncomfortable situation. The MDH research team that are working with this technology needs help to position a transmitter of microwaves to test their equipment. The purpose of this paper is to discover a way to mechanically position a transmitter so that it can be moved along a breast model. The investigation will be made through a product development process in order to review the research question: RQ: “How can a product be designed to position and adjust a microwave transmitter to various locations in order to help testing of cancer research equipment?” By using an agile working methodology in combination with a Design thinking process this thesis includes several sprints that involved continues improvement and feedback from the research team. The first sprint was mostly to discover and experiment on new design ideas as well as control if any of them could work. It resulted in need of measurement changes and redesigning. The second sprint involved measurement corrections. The model itself had the reasonable measurements and the functions worked as expected. However, some of the functions needed to be improved as well as a problem with clearing of the wires to the transmitter itself. The third sprint included changes where more freedom was given and more clearance was made for the wires, but this design turned out to be unpredictable. The fourth sprint included a completely new design to stabilizing the prototype as a result from the researchers’ feedback. To answer the research question, the final design resulted in a 3D printed stand designed to move the transmitter along x-axis as well as rotate around y-axis to adjust to different breast diameters and forms. The stand also includes a rack and pinion design that makes it possible to adjust to different breast lengths. Lastly, the stand makes it possible to gradually move the transmitter around the breast model. However, the final design does not only answer the research question it also fulfils stability and functionality requirements set by the research team. This clarifies why the first iterations needed redesigning. Therefore, the stand is ready for preliminary tests of the researcher’s equipment. To conclude, there are many different design solutions that can answer the research question. However, the design requires stability which reduce the number of design solutions.