Multiview microscopy of single cells through microstructure-based indirect optical manipulation

Fluorescent observation of cells generally suffers from the limited axial resolution due to the elongated point spread function of the microscope optics. Consequently, three-dimensional imaging results in axial resolution that is several times worse than the transversal. The optical solutions to this problem usually require complicated optics and extreme spatial stability. A straightforward way to eliminate anisotropic resolution is to fuse images recorded from multiple viewing directions achieved mostly by the mechanical rotation of the entire sample. In the presented approach, multiview imaging of single cells is implemented by rotating them around an axis perpendicular to the optical axis by means of holographic optical tweezers. For this, the cells are indirectly trapped and manipulated with special microtools made with two-photon polymerization. The cell is firmly attached to the microtool and is precisely manipulated with 6 degrees of freedom. The total control over the cells' position allows for its multiview fluorescence imaging from arbitrarily selected directions. The image stacks obtained this way are combined into one 3D image array with a multiview image processing pipeline resulting in isotropic optical resolution that approaches the lateral diffraction limit. The presented tool and manipulation scheme can be readily applied in various microscope platforms

Integrated optical biosensor for rapid detection of bacteria

In medical diagnostics, rapid detection of pathogenic bacteria from body fluids is one of the basic issues. Most state-of-the-art methods require optical labeling, increasing the complexity, duration and cost of the analysis. Therefore, there is a strong need for developing selective sensory devices based on label-free techniques, in order to increase the speed, and reduce the cost of detection. In a recent paper, we have shown that an integrated optical Mach-Zehnder interferometer, a highly sensitive all-optical device made of a cheap photopolymer, can be used as a powerful lab-on-a-chip tool for specific, labelfree detection of proteins. By proper modifications of this technique, our interferometric biosensor was combined with a microfluidic system allowing the rapid and specific detection of bacteria from solutions, having the surface of the sensor functionalized by bacterium-specific antibodies. The experiments proved that the biosensor was able to detect Escherichia coli bacteria at concentrations of 106 cfu/ml within a few minutes, that makes our device an appropriate tool for fast, label-free detection of bacteria from body fluids such as urine or sputum. On the other hand, possible applications of the device may not be restricted to medical microbiology, since bacterial identification is an important task in microbial forensics, criminal investigations, bio-terrorism threats and in environmental studies, as well

Optikai mikromanipuláció a biofizikában = Optical micromanipulation in biophysics

A projekt új lézercsipesz laboratórium kiépítését finanszírozta. Fő fejlesztés egy fény térmodulátorral (Spatial Light Modulator -SLM) felszerelt lézercsipesz megépítése. Ezzel tetszőlegesen sok csapda független egyidejű programozására van lehetőség. Új litográfiás berendezést is beszereztünk, ezzel mikrofluidikai eszközöket és integrált optikai elemeket készítünk. Az új laboratóriumban új típusú optikai mikromanipulációs kísérleteket végeztünk. Bonyolult alakú teszt objektumokkal összetett mozgásokat lehet megvalósítani. Négy típusú kutatást folytattunk: 1. A torziós manipulációs lehetőséget kihasználva DNS molekula csavarási tulajdonságait viszgáltuk. 2. A fotopolimerizációs struktúra építést és az új lézercsipeszt kihasználva új vizsgálati eszközöket készítettünk, mint mikroviszkozitásmérő, optikai mikromanipulátor. Modellrendszert alkottunk biológiai mozgások modellezésére: Kísérletileg kimutattuk és jellemeztük a hidrodinamikai szinkronizáció jeléenségét. 3. A folyadék mozgatásának fénnyel való vezérlését továbbfejlesztettük, a folyadék áramlási mintázatának fénnyel való változtatását oldottuk meg mikrofluidikai csatornában. 4. Integrált optikai elemeket készítettünk fotopolimerizációval mikrofluidikai alkalmazásra. Nagy érzékenységű interferometrikus szenzort készítettünk, ezt intermolekuláris reakciók jellemzésére, illetve optoelektronikai logikai áramkörök építésére alkalmaztuk. | The project supported the development of a new optical tweezers laboratory. The main development was the building of optical tweezers based on a Spatial Light Modulator (SLM). With this there is possibility to independently program an arbitrary number of optical traps. We also purchased a new photolythography device, this supportsthe building of microfluidics elements and integrated optical parts. In the new laboratory we performed novel optical manipulation experiments.We can realise complicated motions with test objects of complex shape. We worked on four types of experiments: 1. Using the possibility to rotate the trapped objects, we performed torsional manipulation experiments on DNA molecules. 2. Applying the photopolymerisation technique and the new optical tweezers we developed new experimental methods, like microviscosimeter, optical micromanipulator. We also created a model system to mimic biological motions. We experimentally demonstrated and characterised the phenomenon of hydrodynamic synchronisation. 3. We further developed the optical control of fluid flow: we realised the opticaal change of flow pattern in a microfluidics channel. 4. We built integrated optical elements for microfluidics applications. We built a high sensitivity interferometric sensor, and we used this to follow intermolecular interactions and to create optoelectronic logical circuit elements

Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells

We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and non-specifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation. (C)2015 Optical Society of Americ

Optically Trapped Surface-Enhanced Raman Probes Prepared by Silver Photoreduction to 3D Microstructures

3D microstructures partially covered by silver nanoparticles have been developed and tested for surface-enhanced Raman spectroscopy (SERS) in combination with optical tweezers. The microstructures made by two-photon polymerization of SU-8 photoresist were manipulated in a dual beam optical trap. The active area of the structures was covered by a SERS-active silver layer using chemically assisted photoreduction from silver nitrate solutions. Silver layers of different grain size distributions were created by changing the photoreduction parameters and characterized by scanning electron microscopy. The structures were tested by measuring the SERS spectra of emodin and hypericin. © 2015 American Chemical Society

A cross-reactive plasmonic sensing array for drinking water assessment

The continuous monitoring of remote drinking water purification systems is a global challenge with direct consequences for human and environmental health. Here, we utilise a “nano-tastebud” sensor comprised of eight chemically-tailored plasmonic metasurfaces, for testing the composition of drinking water. Through undertaking a full chemometric analysis of the water samples and likely contaminants we were able to optimise the sensor specification to create an array of suitable tastebuds. By generating a unique set of optical responses for each water sample, we show that the array-based sensor can differentiate between untreated influent and treated effluent water with over 95% accuracy in flow and can detect compositional changes in distributed modified tap water. Once fully developed, this system could be integrated into water treatment facilities and distribution systems to monitor for changes in water composition

Functionalization of polymerized 3D microstructures for biological applications

There is an increasing interest in functionalized complex 3D microstructures with sub micrometer features for micro- and nanotechnology applications in biology. Depending primarily on the material of the structures various methods exist to create functional layers of simple chemical groups, biological macromolecules or metal nanoparticles. Using the state of the art microfabrication technology physical objects with dimensions in the micrometer range can be easily made. Microfabrication via lithography of photosensitive materials using spatially selective light exposure is an active research field that utilizes the relatively easily achievable electromagnetic radiation as a source. Two-Photon Polymerization (TPP) is one of the suitable methods for micro-structural patterning that employs non-linear interaction that can help improve pattern resolution into sub-micrometer scale. In this thesis I explore the functionalization of 3D microstructures made my two photon polymerization and their biological application. 3D microstructures produced by TPP have the potential of using them in conjunction with optical tweezers in biological experiments for precise, localized manipulation or probing of various target objects (proteins, DNA, single cells). A key element in the effective application of these microstructures is the functionalization of their surfaces. In my thesis I introduce protein as well as metal nanoparticle (NP) functionalization of TPP microstructures based on amine-terminated linker molecules. The primary goal of the protein coating on the structures is their further use in indirect manipulation of live cells, while the NP coating was used to achieve metal-enhanced fluorescence (MEF) observation. In the thesis I present examples for both applications

Finite groups and elliptic genera

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