Academic literature on the topic 'Nanotechnology - Fluorescent Bioimaging'

Au cours des dernières années, des efforts importants ont été déployés pour développer des métasurfaces (MSs) électromagnétiques avec la possibilité de changer de manière abrupte les propriétés de la lumière. Ces avancées ont ouvert une nouvelle gamme de possibilités pour contrôler la lumière en utilisant des dispositifs optiques ultra-minces. Dans ce contexte, et plus spécifiquement dans le spectre visible, les applications en bio-imagerie s’avèrent particulièrement intéressantes. Une technique qui est particulièrement bien adaptée à l'étude de molécules proches d'une membrane cellulaire est la microscopie à fluorescence par réflexion interne (TIRFM), qui repose sur un champ évanescent d'excitation. Dans ce cas la lumière incidente est totalement réfléchie sur une interphase (typiquement verre/eau) en raison de son angle d'incidence élevé. À ce jour, la TIRFM est généralement mise en œuvre à l'aide d'objectifs volumineux de grande ouverture numérique et de petit champ de vision.Dans ce travail de thèse, nous réalisons de substrats pour la microscopie TIRF à base de métasurfaces constituées de réseaux périodiques de structures asymétriques fabriquées en dioxyde de titane (TiO2) sur du verre borosilicaté. Ces structures, aussi petites que 48 nm, ont été optimisées à l’aide de simulations numériques "Rigorous coupled-wave analysis” (RCWA) dans le but de coupler de 50 à 90% de la lumière incidente dans le premier ordre de diffraction avec des angles élevés (θ &gt; 63deg). Le fait de pouvoir utiliser des objectifs de faible grossissement et d'avoir une grande zone de champ évanescent fournit des conditions TIRF uniques qui ne sont pas accessibles par les méthodes traditionnelles. De plus, ces structures sont compatibles avec la lithographie par nanoimpression UV, ce qui permet d’envisager une fabrication à bas coût et à grande échelle. Outre la conception, et la fabrication, dans cette thèse nous aboutissons à une preuve de principe de la microscopie TIRF basée sur des métasurfaces en milieu biologique en imageant notamment des membranes fluorescentes de cellules souches. Ces métasurfaces permettent ainsi l’implémentation TIRFM à contraste élevé et à faible photo-blanchissement compatible avec des microscopes à champ large peu coûteux<br>In recent years there has been a significant effort to push electromagnetic metasurfaces with the ability to abruptly change light properties into visible wavelengths. These advancements have opened a new range of possibilities to reshape light using ultra-thin optical devices and there is one field that is starting to gather attention: bioimaging. One technique particularly well suited for the study of molecules near a cell membrane is Total Internal Reflection Fluorescence (TIRF) microscopy, which relies on an evanescence field created by light being totally internally reflected within a glass substrate due to its high incidence angle. As of today, TIRF is generally implemented using bulky high-NA, small field of view oil objectives.In this project we present the realization of metasurface-based TIRF microscopy substrates consisting of periodic 2D arrays of asymmetric structures fabricated in titanium dioxide on borosilicate glass. These patterns, as small as 48nm, were optimized through rigorous coupled-wave analysis to couple 50-90% of the incoming normally incident light into the first diffraction order, which outputs at an angle that suffices total internal reflection in water and eliminates the requirement for high NA objectives or prisms to achieve TIRF. Being able to utilize lower-magnification air objectives and having a large evanescence field area provide unique TIRF conditions not accessible by traditional methods. Additionally, these structures are compatible with soft UV nanoimprint lithography, for cost-effective scale production, to give TIRF’s high contrast, low photodamage and low photobleaching capabilities to inexpensive wide-field microscopes

The American Cancer Society predicts that 1,665,540 people will be diagnosed with cancer, and 585,720 people will die from cancer in 2014. One of the most common types of cancer in the United States is skin cancer. Melanoma alone is predicted to account for 10,000 of the cancer related deaths in 2014. As a highly mobile and aggressive form of cancer, melanoma is difficult to fight once it has metastasized through the body. Early detection in such varieties of cancer is critical in improving survival rates in afflicted patients. Present methods of detection rely on visual examination of suspicious regions of tissue via various forms of biopsies. Accurate assessment of cancerous cells via this method are subjective, and often unreliable in the early stages of cancer formation when only few cancer cells are forming. With fewer cancer cells, it is less likely that a cancer cell will appear in a biopsied tissue. This leads to a lower detection rate, even when cancer is present. This lack of detection when cancer is in fact present is referred to as a false negative. False negatives can have a highly detrimental effect on treating the cancer as soon as possible. More accurate methods of detecting cancer in early stages, in a nonsubjective form would alleviate these problems. A proposed alternative to visual examination of biopsied legions is to utilize fluorescent nanocrystalline biomarker constructs to directly attach to the abnormal markers found on cancerous tissues. Quantum dots (QDs) are hydrophobic nanoscale crystals composed of semiconducting materials which fluoresce when exposed to specific wavelengths of radiation, most commonly in the form of an ultraviolet light source. The QD constructs generated were composed of cadmium-selenium (CdSe) cores encapsulated with zinc-sulfide (ZnS) shells. These QDs were then encapsulated with phospholipids in an effort to create a hydrophilic particle which could interact with polar fluids as found within the human body. The goal of this thesis is to develop a method for the solubilization, encapsulation, and initial functionalization of CdSe/ZnS QDs. The first stage of this thesis focused on the generation of CdSe/ZnS QDs and the fluorescence differences between unshelled and shelled QDs. The second stage focused on utilizing the shelled QDs to generate hydrophilic constructs by utilizing phospholipids to bind with the QDs. Analysis via spectroscopy was performed in an effort to characterize the difference in QDs both prior to and after the encapsulation process. The method generated provides insight on fluorescence trends and the encapsulation of QDs in polar substances. Future research focusing on the repeatability of the process, introducing the QD constructs to a biological material, and eventual interaction with cancer cells are the next steps in generating a new technique to target and reveal skin cancer cells in the earliest possible stages without using a biopsy.

Fluorescence microscopy is an essential tool in biomedical sciences that allows specific molecules to be visualized in the complex and crowded environment of cells. The continuous introduction of new imaging techniques makes microscopes more powerful and versatile, but there is more than meets the eye. In addition to develop- ing new methods, we can work towards getting the most out of existing data and technologies. By harnessing unused potential, this work aims to increase the richness, reliability, and power of fluorescence microscopy data in three key ways: through standardization, evaluation and innovation. A universal standard makes it easier to assess, compare and analyze imaging data – from the level of a single laboratory to the broader life sciences community. We propose a data-standard for fluorescence microscopy that can increase the confidence in experimental results, facilitate the exchange of data, and maximize compatibility with current and future data analysis techniques. Cutting-edge imaging technologies often rely on sophisticated hardware and multi-layered algorithms for reconstruction and analysis. Consequently, the trustworthiness of new methods can be difficult to assess. To evaluate the reliability and limitations of complex methods, quantitative analyses – such as the one present here for the 3D SPEED method – are paramount. The limited resolution of optical microscopes prevents direct observation of macro- molecules like DNA and RNA. We present a multi-color, achromatic, cryogenic fluorescence microscope that has the potential to produce multi-color images with sub-nanometer precision. This innovation would move fluorescence imaging beyond the limitations of optics and into the world of molecular resolution.