Micro- and nanotechnology for cell biophysics

Procedures and methodologies used in cell biophysics have been improved tremendously with the revolutionary advances witnessed in the micro- and nanotechnology in the last two decades. With the advent of microfluidics it became possible to reduce laboratory-sized equipment to the scale of a microscope slide allowing massive parallelization of measurements with extremely low sample volume at the cellular level. Optical micromanipulation has been used to measure forces or distances or to alter the behavior of biological systems from the level of DNA to organelles or entire organisms. Among the main advantages is its non-invasiveness, giving researchers an invisible micro-hand to “touch” or “feel” the system under study, its freely and very often quickly adjustable experimental parameters such as wavelength, optical power or intensity distribution. Atomic force microscopy (AFM) opened avenues for in vitro biological applications concerning with single molecule imaging, cellular mechanics or morphology. As it can operate in liquid environment and at human body temperature, it became the most reliable and accurate nanoforce-tool in the research of cell biophysics. In this paper we review how the above three techniques help increase our knowledge in biophysics at the cellular level

Light Sailboats: Laser driven autonomous microrobots

We introduce a system of light driven microscopic autonomous moving particles that move on a flat surface. The design is simple, yet effective: Micrometer sized objects with wedge shape are produced by photopolymerization, they are covered with a reflective surface. When the area of motion is illuminated perpendicularly from above, the light is deflected to the side by the wedge shaped objects, in the direction determined by the position and orientation of the particles. The momentum change during reflection provides the driving force for an effectively autonomous motion. The system is an efficient tool to study self propelled microscopic robots

Holographic multi-focus 3D two-photon polymerization with real-time calculated holograms.

Two-photon polymerization enables the fabrication of micron sized structures with submicron resolution. Spatial light modulators (SLM) have already been used to create multiple polymerizing foci in the photoresist by holographic beam shaping, thus enabling the parallel fabrication of multiple microstructures. Here we demonstrate the parallel two-photon polymerization of single 3D microstructures by multiple holographically translated foci. Multiple foci were created by phase holograms, which were calculated real-time on an NVIDIA CUDA GPU, and displayed on an electronically addressed SLM. A 3D demonstrational structure was designed that is built up from a nested set of dodecahedron frames of decreasing size. Each individual microstructure was fabricated with the parallel and coordinated motion of 5 holographic foci. The reproducibility and the high uniformity of features of the microstructures were verified by scanning electron microscopy

Phase measurement using DIC microscopy

The development of fluorescent probes and proteins has helped make light microscopy more popular by allowing the visualization of specific subcellular components, location and dynamics of biomolecules. However, it is not always feasible to label the cells as it may be phototoxic or perturb their functionalities. Label-free microscopy techniques allow us to work with live cells without perturbation and to evaluate morphological differences, which in turn can provide useful information for high-throughput assays. In this study, we use one of the most popular label-free techniques called differential interference contrast (DIC) microscopy to estimate the phase of cells and other nearly transparent objects and instantly estimate their height. DIC images provide detailed information about the optical path length (OPL) differences in the sample and they are visually similar to a gradient image. Our previous DIC construction algorithm outputs an image where the values are proportional to the OPL (or implicitly the phase) of the sample. Although the reconstructed images are capable of describing cellular morphology and to a certain extent turn DIC into a quantitative technique, the actual OPL has to be computed from the input DIC image and the microscope calibration settings. Here we propose a computational method to measure the phase and approximate height of cells after microscope calibration, assuming a linear formation model. After a calibration step the phase of further samples can be determined when the refractive indices of the sample and the surrounding medium is known. The precision of the method is demonstrated on reconstructing the thickness of known objects and real cellular samples

Mikromanipulációs kísérletek lézercsipesszel = Micromanipulation experiments with optical tweezers

A kutatás az optikai mikromanipuláció területén, különböző irányokban végzett fejlesztéseket és kutatási alkalmazásokat képviselt. A mikromanipuláció területén azt vizsgáltuk, milyen új manipulációs lehetőségeket nyújtanak speciális alakú próbatestek (ellentétben az általáéban használt gömb alakkal). Ennek során kidolgoztuk a lézeres fotopolimerizációs struktúra építés technikáját. Ezzel egyrészt a mikromanipuláció új lehetőségeit vizsgáltuk. Például, lehetőség nyílik torziós manipulálásra, csavarásra, meghatároztuk a DNS molekula torziós rugalmasságát. Ezen kívül, bonyolult struktúrákat építettünk, fénnyel hajtott mikrogépeket, mikrofluidikai csatornákat, integrált optikai szenzorokat. Ezekből bonyolultab összetett rendszereket raktunk össze: fénnyel vezérelt fluorescencia aktivált optikai sejtszeparátort. Kidolgoztuk a fénnyel vezérelt elektroozmózis eljárását. Itt elektromos térrel mozgatott folyadék áramlását vezéreljük fénnyel, ez érdekes jelenség, és új lehetőségeket nyújt mikrofluidikai rendszerek vezérlésében. | The research represented studies in the area of optical micromanipulation, both developing new procedures and basic applications. In the area of micromanipulation we investigated, what new possibilities are offered by test objects of special shapes (as opposed to the generally used spheres). In this process we developed the structure building by laser induced photopolymerization. With this we studied new possibilities of optical manipulation. For example, a poissibility emerges for torsional manipulation, twisting, we determined the torsional elasticity of DNA molecules. In addition, we built complex structures, light driven micromachines, microfluidics channels, integrated optical sensors. From this we constructed complex systems, e.g. a fluorescence activated cell separator. We developed the method of optically controlled electroosmosis. Here the fluid is driven by electric field and this is controlled by light. This is an interesting phenomenon, and is opens new possibilities in the control of microfluidics systems

Three-dimensional femtosecond laser processing for lab-on-a-chip applications

AbstractThe extremely high peak intensity associated with ultrashort pulse width of femtosecond laser allows us to induce nonlinear interaction such as multiphoton absorption and tunneling ionization with materials that are transparent to the laser wavelength. More importantly, focusing the femtosecond laser beam inside the transparent materials confines the nonlinear interaction only within the focal volume, enabling three-dimensional (3D) micro- and nanofabrication. This 3D capability offers three different schemes, which involve undeformative, subtractive, and additive processing. The undeformative processing preforms internal refractive index modification to construct optical microcomponents including optical waveguides. Subtractive processing can realize the direct fabrication of 3D microfluidics, micromechanics, microelectronics, and photonic microcomponents in glass. Additive processing represented by two-photon polymerization enables the fabrication of 3D polymer micro- and nanostructures for photonic and microfluidic devices. These different schemes can be integrated to realize more functional microdevices including lab-on-a-chip devices, which are miniaturized laboratories that can perform reaction, detection, analysis, separation, and synthesis of biochemical materials with high efficiency, high speed, high sensitivity, low reagent consumption, and low waste production. This review paper describes the principles and applications of femtosecond laser 3D micro- and nanofabrication for lab-on-a-chip applications. A hybrid technique that promises to enhance functionality of lab-on-a-chip devices is also introduced

Direct writing of optical microresonators in a lab-on-a-chip for label-free biosensing

Whispering gallery mode (WGM) resonators are promising optical structures for microfluidic label-free bio-sensors mainly due to their high sensitivity, but from a practical point of view they present numerous constraints that make their use in real laboratory diagnosis application difficult. Herein we report on a monolithic lab on a chip fabricated by a hybrid femtosecond laser micromachining approach, for label-free biosensing. It consists of a polymer WGM microresonator sensor integrated inside a glass microfluidic chip, presenting a refractive index change sensitivity of 61 nm per RIU. The biosensing capabilities of the device have been demonstrated by exploiting the biotin-streptavidin binding affinity, obtaining a measurable minimum surface density increase of 67 x 10(3) molecules per mu m(2)