Innovations of wide-field optical-sectioning fluorescence microscopy : toward high-speed volumetric bio-imaging with simplicity

Optical microscopy has become an indispensable tool for biological researches since its invention, mostly owing to its sub-cellular spatial resolutions, non-invasiveness, instrumental simplicity, and the intuitive observations it provides. Nonetheless, obtaining reliable, quantitative spatial information from conventional wide-field optical microscopy is not always intuitive as it appears to be. This is because in the acquired images of optical microscopy the information about out-of-focus regions is spatially blurred and mixed with in-focus information. In other words, conventional wide-field optical microscopy transforms the three-dimensional spatial information, or volumetric information about the objects into a two-dimensional form in each acquired image, and therefore distorts the spatial information about the object. Several fluorescence holography-based methods have demonstrated the ability to obtain three-dimensional information about the objects, but these methods generally rely on decomposing stereoscopic visualizations to extract volumetric information and are unable to resolve complex 3-dimensional structures such as a multi-layer sphere.

The concept of optical-sectioning techniques, on the other hand, is to detect only two-dimensional information about an object at each acquisition. Specifically, each image obtained by optical-sectioning techniques contains mainly the information about an optically thin layer inside the object, as if only a thin histological section is being observed at a time. Using such a methodology, obtaining undistorted volumetric information about the object simply requires taking images of the object at sequential depths.

Among existing methods of obtaining volumetric information, the practicability of optical sectioning has made it the most commonly used and most powerful one in biological science. However, when applied to imaging living biological systems, conventional single-point-scanning optical-sectioning techniques often result in certain degrees of photo-damages because of the high focal intensity at the scanning point. In order to overcome such an issue, several wide-field optical-sectioning techniques have been proposed and demonstrated, although not without introducing new limitations and compromises such as low signal-to-background ratios and reduced axial resolutions. As a result, single-point-scanning optical-sectioning techniques remain the most widely used instrumentations for volumetric imaging of living biological systems to date.

In order to develop wide-field optical-sectioning techniques that has equivalent optical performance as single-point-scanning ones, this thesis first introduces the mechanisms and limitations of existing wide-field optical-sectioning techniques, and then brings in our innovations that aim to overcome these limitations. We demonstrate, theoretically and experimentally, that our proposed wide-field optical-sectioning techniques can achieve diffraction-limited optical sectioning, low out-of-focus excitation and high-frame-rate imaging in living biological systems. In addition to such imaging capabilities, our proposed techniques can be instrumentally simple and economic, and are straightforward for implementation on conventional wide-field microscopes. These advantages together show the potential of our innovations to be widely used for high-speed, volumetric fluorescence imaging of living biological systems.

An annotated bibliography of research on coarse and salmonid fish (excluding salmon and trout) found in fresh water in Scotland

The authors provide an extensive annotated bibliography to a full list of species occurring in Scotland, to highlight what is known about them and to indicate potential areas for further research. The list of references brings together published research papers and numerous unpublished theses and reports, including experimental and laboratory studies conducted in Scotland, although some may not have unique application to the fish fauna in Scottish waters. There has been no attempt to include references that are made incidentally in the general literature intended for naturalists.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

The Fin Whale (Balaenoptera physalus L.): a bibliography

Compiled by Patricia Ann Skaptason. "This bibliography is designed to bring together literature on the fin whale, published from 1940 through 1970. newspaper articles, legal matgerial (except that by the International Whaling Commission), biochemical studies, juvenile and strictly narrative works have been omitted"

A bibliography of the rivers North Tyne, Wear, Tees and Swale

This bibliography covers published and unpublished work on the freshwater sections of the rivers North Tyne, Wear, Tees and Swale, their catchment areas and their tributaries. 393 references are included in the bibliography.

A bibliography of samplers for benthic invertebrates

This annotated bibliography covers literature to the end of November 1977, and includes references to samplers that could be used for the rapid removal of benthic invertebrates from natural substrata of rivers and streams. Marine samplers which have been, or could be, used in freshwater. Coverage of Russian literature is incomplete, although a selection of recent and important references are included. The references are arranged under the following headings, Reviews; Nets and quadrat samplers; Scoops, shovels and dredges; Grabs; Corers; Suction and air-lift samplers; Electroshocking samplers; Efficiencies and comparisons; and Samplers from catalogues. There is an index to samplers (by the common name) and an author index.

A bibliography of works for the identification of freshwater invertebrates in the British Isles

This bibliography covers the literature up to the end of 1978. The criteria used in the selection of references were that they should aid identification of invertebrates directly; thus, works solely concerned with the taxonomy of a particular group are in general omitted unless they contain a key. Some check-lists are however included where they give current nomenclature. The references are arranged alphabetically within each group and deal mainly with macro-invertebrates but include available keys to some microscopic invertebrates. Internal parasites and hymenopterous parasitoids are omitted. For insects the life stages to which the key applies are given where this is not clear in the reference. A number of keys to non-aquatic stages have been included in the hope that they may prove useful in certain circumstances. In addition, under a general head, latest check-lists are referred to together with bibliographies of algal keys and a guide for the identification of British water plants.

Petroleum hydrocarbons in fresh waters: a preliminary desk study and bibliography

A literature survey was carried out into the effects of petroleum hydrocarbons in freshwater, from the toxicity, biodegradability and concentration aspects. It was supplemented by a selective search on hydrocarbons in the marine environment for comparison. The aim was to determine the major inputs of these hydrocarbons, their accumulation, effects and fate in freshwaters. The search was confined to the period 1965-1978. The bibliography contains 390 references, divided by subject.

An annotated bibliography of aquatic sediment traps and trapping methods

This annotated bibliography is intended to give as reasonably complete a review of the existing literature as possible, and to offer some practical guidance in the selection and operation of sediment traps in future monitoring programmes.

Valoración de bio-oil completo por craqueo catalítico en condiciones de la unidad FCC de refinería

351 p.

A supplement to a bibliography of samplers for benthic invertebrates

A supplement to the earlier bibliography compiled by Elliott and Tullett 1978 (FBA Occas. Publ. No. 4) covering literature from December 1977 - December 1982 on samplers that could be used for the rapid removal of benthic intertebrates from the natural substrata of rivers and streams. In addition it includes papers on marine samplers that have been or could be used in freshwater.

An indexed bibliography of papers on tagging of tunas and billfishes

Two working parties, the Working Party on Tuna Tagging in the Pacific and Indian Oceans and the Working Party on Tuna Tagging in the Atlantic and Adjacent Seas, were formed by the Food and Agriculture Organization (FAD) of the United Nations in 1966 (Anonymous, 1966c). The conveners of these working parties were Dr. James Joseph of the Inter-American Tropical Tuna Commission (IATTC) and Mr. FrankJ. Mather, III, of the Woods Hole Oceanographic Institution (WHOI). In 1969 it was recommended that the working parties direct their attention toward billfishes, as well as tunas (Anonymous, 1969h: 5). One report (Joseph and working party, 1969) was published by the Pacific and Indian Oceans group and two (Mather and working party, 1969 and 1972) were published by the Atlantic Ocean and adjacent seas group. Each of the three working party reports included a bibliography of tuna and billfish tagging. The compiler of this bibliography, beginning in 1970, prepared numerous memoranda to the members of the working party, most of which included lists of papers on tuna and billfish tagging which had come to his attention, either directly or through members of the working party. The bibliographies in the three working party reports and the lists of references in the memoranda form the basis for the present bibliography.

Poly(lactic acid)-based bio-blends and nanocomposites

172 p.

Avaliação da acidez de bio-óleos obtidos pela pirólise de óleos vegetais pós-consumo

O descarte irregular do óleo vegetal pós-consumo diretamente na rede de esgoto vem causando grandes problemas para o meio ambiente. Atualmente, essa problemática tem se intensificado devido ao aumento de produção e consumo destes óleos, o que por conseqüência aumenta o despejo desordenado. No presente estudo foi realizada a obtenção do bio-óleo a partir da pirólise térmica em atmosfera de nitrogênio, a 400C, por 20 minutos, do óleo vegetal pós-consumo, provenientes das seguintes oleoginosas: soja, milho, girassol e canola. As pirólises não-catalíticas apresentaram uma geração média entre 40 e 50% de um bio-óleo, de elevado índice de acidez, 81,8 mg NaOH/g. Na pirólise catalítica, a argila ácida K10 foi o catalisador que apresentou melhor eficácia para geração de um bio-óleo de menor índice de acidez. A concentração ótima do catalisador foi de 5%(m/m), gerando 482 % de um bio-óleo com índice de acidez de 43,8 mg NaOH/g. A caracterização dos líquidos pirolíticos obtidos foi realizada através da técnica de espectrofotometria na região do infravermelho (FTIR) e cromatografia em fase gasosa acoplada a espectrômetro de massas (CG/EM) que monstraram que a trioleína, o triglicerídeo do ácido oléico, foi craqueado, gerando o hexadecanoato de octadecila e o oleato de eicosila, ésteres do respectivo ácido graxo