M.Ing. (Electrical and Electronic Engineering) / Advances in bio-imaging have triggered the development of a highly sophisticated imaging tool known as fluorescence microscopy. Fluorescence microscopy is used in many biological applications to visualize sub-cellular processes and gives the ability to image three-dimensional (3D) structures in living cells. The use of fluorescence microscopy and specific staining methods make biological molecules appear as bright spots in image data. The analysis of fluorescence microscopy images requires the detection and tracking of hundreds spots in image data and is of great importance for biologists to better understand cell functions. However, the analysis of these data is still performed manually in most biological laboratories worldwide. The manual analysis of these data is both time consuming, laborious and susceptible to human errors. Several computer-based algorithms have been proposed for the detection and tracking of spots in microscopy images. Most of these methods were validated on limited image data and relatively few studies have been performed for the comparison of these methods in real applications. This study quantitatively compared the performance of four detection and two tracking methods applied in microscopy images for the analysis of bright spots. The performance of the algorithms was validated on both synthetic and real images. The synthetic images offered a better way of validating algorithms against ground truth reference results. Results indicate that there are major differences in algorithm performance for both detection and tracking. In the detection results the Isotropic Undecimated Wavelet Transform (IUWT) and the Laplacian of Gaussian (LoG) achieved better results than the other methods in comparison when values are considered. The tracking results indicate that the Interacting Multiple Model (IMM) method achieved better results than the Feature Point Tracking (FPT) method when Jaccard Similarity Scores (JSC) are considered.

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Thesis (M.S.E.C.E.)--University of Delaware, 2007. / Principal faculty advisors: Fouad Kiamilev and Robert F. Rogers, Dept. of Electrical and Computer Engineering. Includes bibliographical references.

As a high-resolution wide-field near-surface microscopy, total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules. Unlike those costly, sample consuming and time consuming traditional detection assays, the application of TIRFM enable the direct quantification of biomolecules in a sample pretreatment and enrichment free fashion. Taking advantages of the TIRFM imaging system, in this thesis we have applied the TIRFM imaging system to directly quantify the content of different cancer associated biomarkers. Four different detection approaches for direct cancer biomarkers quantification with the aid of TIRFM were herein presented respectively. In Chapter 2, a direct quantification of nasopharyngeal carcinoma associated miRNAs was described. In the assay, five different miRNAs were chosen as the target analytes, which hybridized with the synthetic complementary LNA, probe in solution. The duplex was labeled with intercalating fluorescence dye YOYO-1 and the signal was then detected by the TIRFM-EMCCD imaging system. The LNA probe exhibited a high binding affinity towards the complementary target miRNAs and a limit of detection of 8 pM was achieved. Since the LOD is far below the reported concentration of miRNAs found in body fluids, this developed assay is of high potential to serve as a tool for non-invasive detection of miRNAs for early disease diagnosis. In Chapter 3, an advanced single-molecule based assay for direct circulating miRNAs detection was developed. The assay was demonstrated to be capable of differentiating the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, locked nucleic acid modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 μL of serum and the whole assay can be completed in an hour. The experimental results agreed well with reported and while the quantity of mir-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising non-invasive detection tool for early NPC diagnosis, monitoring and staging. In chapter 4, a self-assembled protein nanofibril based online pre-concentrating sensor was developed. This solution-based hybridization assay was applied to quantified the amount of target miRNAs, mir-196a. Biotinylated locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The target hybridization duplex was immobilized on the backbone of the nanofibril through the biotin-streptavidin interaction. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient’s serums was also demonstrated. In chapter 5, a magnetic nanoparticles based sandwich immunosensor with carbazole-based cyanine as the fluorescence labeling dye for the direct quantification of prostate cancer related antigen, PSA, was developed. Taking benefit of the magnetic property of the nanoparticles, the target sandwich immunocomposites can be easily online separated from the sample matrix. The as-developed assay can efficiently discriminate the target PSA from other disease related antigens and achieve a LOD of 400 fM (13 pg/mL) and a LOQ of 2 pM (0.66 ng/mL). As the whole detection assay can be completed in 1 h with only 10 μL of sample, this assay is fast and cost effective and of high potential for early disease and cancer diagnosis, staging and monitoring

Thesis (M.S.)--University of Wisconsin--Madison, 1966. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 77-84.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Quantitative three dimensional maps of cellular structure, activity and function provide the key to answering many prevalent questions in modern biological research. Fluorescence microscopy has emerged as an indispensable tool in generating such maps, but common techniques are limited by fundamental physical constraints which render them incapable of simultaneously achieving high spatial and temporal resolution. This thesis will describe the development of novel microscopy techniques and complementary computational tools capable of addressing some of the aforementioned limitations of fluorescence microscopy and further outline their application in providing novel biological insights. The first section details the design of a light sheet microscope capable of high-throughput imaging of cleared, macroscopic samples with cellular resolution. In light sheet microscopy, the combination of spatially confined illumination with widefield detection enables multi-megapixel acquisition in a single camera exposure. The corresponding increase in acquisition speed enables systems level biological studies to be performed. The ability of this microscope to perform rapid, high-resolution imaging of intact samples is demonstrated by its application in a project which established a niche and hierarchy for stem cells in the adult nervous system. Light sheet microscopy achieves fast volumetric imaging rates, but the two dimensional nature of each measurement results in an inevitable lag between acquisition of the initial and final planes. The second section of this thesis describes the development and optimization of a light field microscope which captures volumetric information in a snapshot. Light field microscopy is a computational technique and images are reconstructed from raw data. Both the fidelity of computed volumes and the efficiency of the algorithms are strongly dependent on the quality of the rectification. A highly accurate, automated procedure is presented in this section. Light field reconstruction techniques are investigated and compared and the results are used to inform the re-design of the microscope. The new optical configuration is demonstrated to minimize the long-object problem. In the final section of the thesis, the spatial resolution limits of light field microscopy are explored using a combination of simulations and experiments. It is shown that light field microscopy is capable of localizing point sources over a large depth of field with high axial and lateral precision. Notably, this work paves the way towards frame rate limited super resolution localization microscopy with a depth of field larger than the thickness of a typical mammalian cell.

Two-photon microscopy is often used in biological imaging due to its optical sectioning and depth penetration capabilities. These characteristics have made two-photon microscopy especially useful for neurobiological studies where imaging a volume at single cell resolution is typically required. This dissertation focuses on the optimization of two-photon excited fluorescence for volumetric imaging of biological samples, with special attention to imaging the mouse brain. Chapter 2 studies wavefront manipulation as a way of optimizing two-photon excited fluorescence. We show, through numerical simulations and experiments, that the magnitude of the two-photon fluorescence signal originating from cell-sized objects can be used as a metric of beam quality. We also show that the cranial window used in mouse experiment is a major source of aberrations, which can readily be represented in the Zernike basis. Finally, we implement a modal wavefront optimization scheme that optimizes the wavefront based entirely on the magnitude of the fluorescence. Along with this scheme, Zernike functions are found to be a useful basis for correcting aberrations encountered in mouse brain imaging while the Hadamard basis is found to be useful for scattering compensation. Corrections performed in mouse brain using Zernike functions are found to be valid over hundreds of microns, allowing a single correction to be applied to a whole volume. Finally, we show that the wavefront correction system can double as a wavefront encoding system for experiments that require custom point-spread-functions. Chapter 3 aims to significantly improve the volume imaging rate of two-photon microscopy. The imaging speed is improved by combining two-photon excitation with scanning confocally-aligned planar excitation microscopy (SCAPE). Numerical simulations, analytical arguments, and experiments reveal that the standard method of combining nano-joule pulses with 80 MHz repetition rates is inadequate for two-photon light-sheet excitation. We use numerical simulations and experiments to explore the possibility of achieving fast volumetric imaging using line and sheet excitation and find that the sheet excitation scheme is more promising. Given that two-photon excitation requires high photon-flux-densities near the focus, achieving high enough fluorescence has to be balanced with restrictions placed by saturation, photodamage, photobleaching and sample heating effects. Finally, we experimentally study light sheet excitation at various pulse repetition rates with femtosecond pulses and find that repetition rates near 100 kHz allow imaging of nonbiological samples of ~200x300x300 μm^3 volume at 20 volumes per second while balancing the above constraints. This work paves the way for achieving fast, volumetric two-photon imaging of the mouse brain.