Optical Measurement of Electromechanical Characteristics of Heart Cells

Disertační práce se zabývá aplikací optických metod měření s využitím technik optické mikroskopie a fluorescenční mikroskopie při měření elektromechanických projevů izolovaných srdečních buněk a shluků diferencovaných kardiomyocytů. První navržená metoda využívá účelné kombinace fluorescenční mikroskopie s využitím rychlé fluorescenční kamery s vysokým rozlišením a mikroskopie atomárních sil pro současné měření vápníkových transientů a kontrakce shluků srdečních buněk. Získané signály prochází filtrací, zpracováním a analýzou. Výsledně získané funkční parametry kardiomyocytů získané analýzou signálů po aplikaci kofeinu jsou hodnoceny srovnáním s funkčními parametry získanými při kontrolním měření. Druhá navržená metoda je aplikována na shluky kardiomyocytů za účelem měření jejich kontrakce. Signály získané optickou metodou jsou analyzovány a výsledky porovnány s referenčním signálem získaným měřením s pomocí mikroskopie atomárních sil. Pro spolehlivější a stabilnější měření kontrakce v experimentech zaměřených na současné měření s vápníkovými transienty u izolovaných kardiomyocytů byly navrženy a realizovány optické metody měření kontrakce založené na detekci konců s využitím úpravy mikroskopického obrazu přeostřením a s využitím fluorescenční metody měření.Dissertation is focused on the application of optical measurement methods using techniques of optical microscopy and fluorescence microscopy in measurements of electromechanical characteristics of isolated cardiac cells and clusters of differentiated cardiomyocytes. The first proposed method uses a practical combination of fluorescence microscopy equipped with fluorescent fast and high-resolution camera and atomic force microscopy for simultaneous measurement of calcium transients and contraction of cardiomyocyte clusters. The signals obtained undergoes filtration, processing and analysis. Result function parameters obtained by analyzing signals after application of caffeine are evaluated by comparison with functional parameters obtained during the control measurement. The second proposed method is applied to the cardiomyocyte clusters for the purpose of cardiomyocyte contraction signals measurement. The signals obtained by optical methods are analyzed and compared with the reference signal obtained using atomic force microscopy. Optical measurement method of cell contractins based on detection of cell ends using adjusting of microscopy images by re-sharpening and fluorescence method for cardiomyocyte contractions measurements were designed to increase realiability in simultaneous measurement of cell contractions simultaneously with calcium transients in isolated cardiomyocytes experiments.

Hippocampal Neurons’ Alignment on Quartz Grooves and Parylene Cues on Quartz Substrate

Alignment and patterning of neurons have great importance in some research fields, especially for regenerative medicine and for the formation of artificial neural networks. Alignment of neurons on quartz grooves and parylene cues on quartz substrate was evaluated in this work. The neurons’ alignment on quartz grooves is considered to be topographical alignment, while the neurons’ alignment on parylene cues on quartz substrate is considered to be chemical alignment. Both quartz grooves’ and parylene cues’ widths were fabricated in a range from 2 µm to 8 µm; quartz grooves’ heights were in a range from 0.25 µm to 4 µm, while parylene cues’ heights were only 0.25 µm. Neurons were dissociated hippocampal neurons from rat E18. Neurons were cultivated on test substrates for 7 days before alignment evaluation. As expected, neurons aligned according to the direction of grooves and cues; however, transversal growth direction was also observed with much smaller tendency. Chemical alignment was found to be more effective than topographical alignment. If parylene cues are thin and distanced enough, then neurons have a tendency to follow the direction of individual parylene cues; however, neurons on quartz grooves have a tendency just to follow a preferable direction than individual quartz grooves

The Effect of Rhodamine-Derived Superparamagnetic Maghemite Nanoparticles on the Motility of Human Mesenchymal Stem Cells and Mouse Embryonic Fibroblast Cells

Nanoparticles have become popular in life sciences in the last few years. They have been produced in many variants and have recently been used in both biological experiments and in clinical applications. Due to concerns over nanomaterial risks, there has been a dramatic increase in investigations focused on safety research. The aim of this paper is to present the advanced testing of rhodamine-derived superparamagnetic maghemite nanoparticles (SAMN-R), which are used for their nontoxicity, biocompatibility, biodegradability, and magnetic properties. Recent results were expanded upon from the basic cytotoxic tests to evaluate cell proliferation and migration potential. Two cell types were used for the cell proliferation and tracking study: mouse embryonic fibroblast cells (3T3) and human mesenchymal stem cells (hMSCs). Advanced microscopic methods allowed for the precise quantification of the function of both cell types. This study has demonstrated that a dose of nanoparticles lower than 20 microg-cm -2 per area of the dish does not negatively affect the cells’ morphology, migration, cytoskeletal function, proliferation, potential for wound healing, and single-cell migration in comparison to standard CellTracker Green CMFDA (5-chloromethylfluorescein diacetate). A higher dose of nanoparticles could be a potential risk for cytoskeletal folding and detachment of the cells from the solid extracellular matrix

Upgrade of The Langendorff Apparatus Using The Infrared Thermo-Control System and An Intelligent Heater

Biological experiments involving isolated organs and tissues demand precise temperature monitoring and regulation. An automatic temperature control system was proposed and optimised on real isolated swine hearts and the prototype is described in this work. The traditional Langendorff apparatus consists of a heart holder, a reservoir of perfusion solution flowing to aortic cannula and a heating bath allowing passive heat transfer to the reservoir of perfusion solution. The commercial infrared camera FLIR T62101 was added to this basic set-up and used for very precise monitoring of the temperature kinetic of the organ and connected with an electronic feedback loop, which allowed real-time and precise regulation of heat transfer from the heating bath to the perfusion solution and in turn indirectly to the heart tissue. This provides real time control and active regulation of the myocardial tissue temperature. The infrared camera was tested in several modes and several variants of detection were optimised for ideal measurement of the region of interest of the ex vivo organ. The kinetics of the temperature changes and temperature stability of the tissue were recorded and calibrated by external electronic thermometers (type Pt100, inserted in tissue). The time lapse from the hang-up of the hypo termed organ (30 °C) until optimal warming (37 °C) was less than eight minutes in the final instrument prototype. The final stability of the 37 °C tissue temperature was approved; the temperature fluctuation of left ventricle tissue was characterised as 36.8 ± 0.5 °C. This upgraded traditional instrument could be used in specific preclinical and clinical transplantation and analytical projects in future

System for cardiomyocyte contraction acquisition and evaluation in LabVIEW

V článku je představeno kompletní hardwarové a softwarové řešení pro snímání, záznam a vyhodnocení kontrakce stimulované izolované srdeční buňky. Systém je realizován v prostředí LabVIEW.V článku je představeno kompletní hardwarové a softwarové řešení pro snímání, záznam a vyhodnocení kontrakce stimulované izolované srdeční buňky. Systém je realizován v prostředí LabVIEW

Automatic image-based method for quantitative analysis of photosynthetic cell cultures

This work deals with an automatic quantitative analysis of photosynthetic cell cultures. It uses images captured by a confocal fluorescent microscope for automatic determination the number of cells in sample containing complex 3D structure of cell clusters. Experiments were performed on the confocal microscope Leica TCS SP8 X. The cell nuclei were stained by SYBR® Green fluorescent DNA binding marker. In the first step we used combination of adaptive thresholding to found out areas where nuclei were located. Proposed segmentation steps allowed reduction of noise and artefacts. Z-axis position was obtained as a location of peak from intensity profile. Finally model of scene can be created by emplacement of spheres with adequate diameter to found 3D coordinates. Number of cells per volumetric unit were determined in structurally different culture samples of cell cultures Chenopodium rubrum (Cr) and Solanum lycopersicum (To). The results were verified by manual counting. © Springer International Publishing Switzerland 2016

A Novel Gesture-Based Control System for Fluorescence Volumetric Data in Virtual Reality

With the development of light microscopy, it is becoming increasingly easy to obtain detailed multicolor fluorescence volumetric data. The need for their appropriate visualization has become an integral part of fluorescence imaging. Virtual reality (VR) technology provides a new way of visualizing multidimensional image data or models so that the entire 3D structure can be intui-tively observed, together with different object features or details on or within the object. With the need for imaging advanced volumetric data, the demands on the control of virtual object prop-erties are increasing, it happens especially for multicolor objects obtained by fluorescent mi-croscopy. Existing solutions with universal VR controllers or software-based controllers with the need to define sufficient space for the user to manipulate data in VR are not usable in many practical applications. Therefore, we developed a custom gesture-based VR control system with a custom controller connected with the FluoRender visualization environment. The multi-touch sensor disc was used for this purpose. Our control system may be a good choice for easier and more com-fortable manipulation of virtual objects and their properties, especially using confocal microscopy, which is the most widely used technique for acquiring volumetric fluorescence data so far