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Focused electron beam induced nanostructures for the calibration of Superresolution Blink-Microscopy

Visser, B. (2013) Focused electron beam induced nanostructures for the calibration of Superresolution Blink-Microscopy. Master's Thesis / Essay, Physics.

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In this research we are creating novel materials by decorating samples locally with functional molecules. The used functional molecules are different types of fluorophores. Fluorophores are used to functionalize SiO features designed as calibration structures for superresolution blink-microscopy with a method called MACE-ID: molecular assembly controlled by electron-beam induced deposition. The role of the height in deposited features has been determined by investigating interference effects on small deposits. In order to establish the best experimental setting for superresolution imaging the relevant parameters concerning sample handling have been investigated including surface activation, the role of the substrate and the means of storage. The role of reducing and oxidizing molecules in the imaging buffer is investigated in EPI. For the fluorescent ATTO 655 label the best signal-to-noise ratio has been achieved in the absence of oxygen using an oxygen scavenging system. The best blinking parameters for the ATTO 655 label in total internal reflection fluorescence (TIRF) mode are established using DNA immobilization on a cover slide. Imaging in TIRF mode of nanostructures deposited on a diamond-like carbon (DLC) coated cover slide is achieved resulting in the best achieved signal-to-noise ratio. This signal-to-noise ratio is high enough in order to detect single emitting molecules. However, all imaged nanostructures are too highly labeled, preventing the temporal separation of fluorophores. To pave the way for sub-diffraction limited imaging of nanostructures, appropriate labeling densities can easily be achieved by optimizing the incubation time and concentration of the dye solution or applying non-fluorescent labels. This reproducible method generating samples with high signal-to-noise ratios gives exciting new possiblities for the creation of robust calibration devices for superresolution microscopy.

Item Type: Thesis (Master's Thesis / Essay)
Degree programme: Physics
Thesis type: Master's Thesis / Essay
Language: English
Date Deposited: 15 Feb 2018 07:55
Last Modified: 15 Feb 2018 07:55

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