A versatile maskless microscope projection photolithography system and its application in light-directed fabrication of DNA microarrays

We present a maskless microscope projection lithography system (MPLS), in which photomasks have been replaced by a Digital Micromirror Device type spatial light modulator (DMD, Texas Instruments). Employing video projector technology high resolution patterns, designed as bitmap images on the computer, are displayed using a micromirror array consisting of about 786000 tiny individually addressable tilting mirrors. The DMD, which is located in the image plane of an infinity corrected microscope, is projected onto a substrate placed in the focal plane of the microscope objective. With a 5x(0.25 NA) Fluar microscope objective, a fivefold reduction of the image to a total size of 9 mm2 and a minimum feature size of 3.5 microns is achieved. Our system can be used in the visible range as well as in the near UV (with a light intensity of up to 76 mW/cm2 around the 365 nm Hg-line). We developed an inexpensive and simple method to enable exact focusing and controlling of the image quality of the projected patterns. Our MPLS has originally been designed for the light-directed in situ synthesis of DNA microarrays. One requirement is a high UV intensity to keep the fabrication process reasonably short. Another demand is a sufficient contrast ratio over small distances (of about 5 microns). This is necessary to achieve a high density of features (i.e. separated sites on the substrate at which different DNA sequences are synthesized in parallel fashion) while at the same time the number of stray light induced DNA sequence errors is kept reasonably small. We demonstrate the performance of the apparatus in light-directed DNA chip synthesis and discuss its advantages and limitations.Comment: 12 pages, 9 figures, journal articl

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized

Nonequilibrium effects in DNA microarrays: a multiplatform study

It has recently been shown that in some DNA microarrays the time needed to reach thermal equilibrium may largely exceed the typical experimental time, which is about 15h in standard protocols (Hooyberghs et al. Phys. Rev. E 81, 012901 (2010)). In this paper we discuss how this breakdown of thermodynamic equilibrium could be detected in microarray experiments without resorting to real time hybridization data, which are difficult to implement in standard experimental conditions. The method is based on the analysis of the distribution of fluorescence intensities I from different spots for probes carrying base mismatches. In thermal equilibrium and at sufficiently low concentrations, log I is expected to be linearly related to the hybridization free energy $\Delta G$ with a slope equal to $1/RT_{exp}$, where $T_{exp}$ is the experimental temperature and R is the gas constant. The breakdown of equilibrium results in the deviation from this law. A model for hybridization kinetics explaining the observed experimental behavior is discussed, the so-called 3-state model. It predicts that deviations from equilibrium yield a proportionality of $\log I$ to $\Delta G/RT_{eff}$. Here, $T_{eff}$ is an effective temperature, higher than the experimental one. This behavior is indeed observed in some experiments on Agilent arrays. We analyze experimental data from two other microarray platforms and discuss, on the basis of the results, the attainment of equilibrium in these cases. Interestingly, the same 3-state model predicts a (dynamical) saturation of the signal at values below the expected one at equilibrium.Comment: 27 pages, 9 figures, 1 tabl

The influence inclination of supply pipe to the Q/H characteristic of measuring flume PARS P3

Tato bakalářská práce se zabývá vlivem přívodního potrubí na Q/H charakteristiku měrného žlabu PARS P3. Byla provedena měření Q/H charakteristik pod různými sklony přívodního potrubí a měrného žlabu. Následně pak byly tyto měření zpracovány a z výsledků byly stanoveny korekce a nejistoty měření.This bachelor thesis deals with the influence of the inlet pipe on the Q / H characteristic of the measuring flume PARS P3. Measurements of Q / H characteristics were performed under different inclination of the inlet pipe and the measuring flume. Subsequently, these measurements were processed and the corrections and uncertainties of the measurements were determined from the results.

Detection, Description and Tracking of Ants in Video Sequences

katedra kybernetik

Tracking, Learning and Detection of Multiple Objects in Video Sequences

Sledování většího počtu objektů ve video sekvencích v sobě zahrnuje mnoho nejrůznějších podproblémů, včetně sledování zvířat během biologických experimentů. Jejich automatické, či poloautomatické vyhodnocení, má pro biology obrovský význam. V této tezi představujeme metodu pro sledování většího počtu objektů v laboratorních podmínkách. Obtíže způsobené možnou ztrátou identity během interakcí, kdy dochází k nepřehledným situacím, jsou řešeny pomocí rozpoznávání identity jedince. Rozšířili jsme aktuálně nejlepší metodu pro klasifikaci okem těžko rozeznatelných objektů a na sekvencích zachycujících mravence, ryby a svinky jsme ukázali, že tímto způsobem lze dosáhnout zlepšení o 4,5-18%. Metodu jsme implementovali a doplnili o uživatelské rozhraní pro zobrazení a editaci výsledků, umožňující interakci a doplnění informací v průběhu výpočtů.The domain of Multi-Object Tracking encompasses various interesting problems including animal surveillance in biology experiments. Automated or semi-automated evaluation of experiments has a tremendous impact in biology. In this thesis, we are introducing a method for tracking of multiple interacting objects in laboratory conditions. The difficulties of identity maintenance arising from object interactions and occlusions are solved by identity re-detection. We have enhanced current state-of-the-art classifier for objects hardly distinguishable by a naked eye, and we have shown that it improves the performance for per region classification by 4.5%-18% on video sequences with ants, zebrafish, and bugs. The method implementation is accompanied with a graphical user interface. It includes tools for manual annotation of difficult object interactions that hint the tracker and also means for final correction of tracking output

Sports hall with facilities and restaurants, Valašské Klobouky

Diplomová práce se týká novostavby samostatně stojící sportovní haly se zázemím a restaurací ve Vlašských Kloboukách. Sportovní hala má jedno podlaží částečně zapuštěné pod terénem, obsahuje tribunu pro 340 diváků. Druhá část, tj. zázemí sportovní haly je v podzemním podlaží, v nadzemním podlaží je restaurace s kuchyní. Objekt je navržen v půdorysném tvaru dvou obdélníků. Střecha je sedlová a pultová. Obvodové konstrukce u haly jsou provětrávané, část se zázemím a restaurací je kontaktně zateplen. Zázemí zaměstnanců restaurace jsou situovány v nadzemním podlaží, strojovny VZT jsou celkem 3, v části haly, v 1S a v 1NP. Pro vytápění je navrhnut výměník, ke kterému je přiveden horkovod. Povrchovou úpravu tvoří keramická dlažba a v hale je použita dvojitá palubovka. Nosnou konstrukci nad 1S tvoří prefabrikované dílce spiroll.This thesis covers brand new detached sports hall with facilities and restaurants in Walnut Klobouky. Sports hall has a floor partly sunk below the surface, contains grandstand for 340 spectators. The second part, ie background sports hall is in the basement, the ground floor is a restaurant kitchen. The building is designed in plan shape of two rectangles. The roof is gabled and desk. Cladding the halls are ventilated, part of the facilities and restaurants are contact-insulated. Staff restaurant facilities are located in the floor, engine room ventilation are 3, in the hall, first underground floor and first floor. The heat exchanger is designed, which is applied hot water. The finish consists of ceramic tiles in the lobby is used double wood board flooring. The supporting structure consists of prefabricated unit first underground floor SPIROLL.

Understanding fractional equivalence and the differentiated effects on operations with fractions

This study compared two representations for teaching fraction equivalence. It traced the implications of both representations on the students comprehension of fractions as well as their ability to perform operations with fractions. The participants in the study included 65 sixth grade students in three extant classrooms. Two classes were instructed using the textbook representation while the third class received instruction using a representation presented by Van de Walle and recommended by the National Council for Teaching Mathematics. Data were collected from pre-tests, post-tests, student work samples, field notes and a semi-structured interview. Qualitative analyses were used to analyze the data. Items were coded for procedural and conceptual understanding and categorized into levels of proficiency. Additionally, items involving operations with fractions were coded for error patterns. Conclusions were drawn about how the different representations affected student comprehension and faculty with fractions