Acerbo, Esteban (2022) Mediciones de impedancia eléctrica espectral en células y desarrollo de un arreglo dual de amplificadores lock-in / Spectral electrical impedance measurements in cells and development of a dual array of lock-in amplifiers. Maestría en Ciencias Físicas, Universidad Nacional de Cuyo, Instituto Balseiro.
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Resumen en español
Durante la realización de este trabajo hemos estudiado la impedancia espectral de electrodos desnudos con respecto a la distancia entre microelectrodos tierra y microelectrodos excitados, observando que la resistencia y capacidad no cambian con la misma. Analizamos la impedancia espectral de distintos microelectrodos, desnudos y con células, observando muy buena repetitividad. Desarrollamos un nuevo método para predecir y estimar el radio de sanación en función del tiempo mediante mediciones no intrusivas de impedancia eléctrica espectral. Aplicamos el modelo Mesoscópico sobre datos experimentales obtenidos por técnica ECIS durante ensayos de herida y cicatrización, pudiendo estimar el radio de sanación en función del tiempo. Al comparar los datos del radio cicatrizado obtenidos por este método con mediciones realizadas a partir de fotos al final del proceso de sanación, se observa un buen acuerdo entre ellos. Para estos ensayos cultivamos una monocapa confluente de células MDCK tipo II sobre un microelectrodo. Luego, sometimos sobre este sistema una corriente suficientemente alta para producir electroporación severa en las células, que se encuentran sobre la superficie del microelectrodo, y finalmente su muerte. Posteriormente se monitoreó la evolución del cultivo a través de su impedancia eléctrica espectral. Aplicamos cuatro algoritmos de cálculo de dimensión fractal sobre la señal temporal de impedancia eléctrica de cultivos celulares MDCK tipo II normales monitoreados por técnica ECIS. Por ´estos se mostró que la dimensión fractal debida al micromovimiento permite discriminar procesos no sensados por la impedancia espectral del mismo. En este trabajo sometimos cultivos celulares a través de daño por radiación ionizante, daño por corriente eléctrica y exposición a fármacos para analizar los cambio en la estructura fractal de la señal temporal. Entre los cambios presentados y detectados en la estructura fractal se encuentra la diferenciación de una monocapa sana y una expuesta a un fármaco, como también la diferenciación entre un proceso de siembra y de cicatrización de herida realizada por corriente eléctrica. Los cuatro algoritmos utilizados fueron validados al aplicarlos sobre funciones topológicas de dimensión fractal conocida, estudio que determinó las condiciones necesarias para una correcta estimación al utilizar datos experimentales. Desarrollamos y ensayamos con impedancias conocidas, un novedoso dispositivo de medición utilizando dos lock-in amplifiers, para excitar un sistema con dos frecuencias simultáneamente. Luego, empleando esta configuración para medir impedancias espectrales de microelectrodos con y sin células. A partir de las mediciones de impedancia eléctrica calculamos la autocorrelación y correlación cruzada para señales, de resistencia y capacidad, realizadas con excitaciones de baja y alta frecuencia. Obtuvimos, en baja frecuencia, tiempos de correlación de 20 segundos y evidencia de ruido blanco en alta frecuencia. Simulamos numéricamente resultados del uso de dos lock-in’s y el efecto de implementar filtros de distintos órdenes. Realizamos ensayos de radiosensibilidad sobre cultivos MDCK tipo II normales monitoreando el estado celular por técnica ECIS. En este trabajo irradiamos cultivos celulares en estado confluente administrando 5, 15 y 17 Gy en distintas ocasiones. No observamos cambios en los valores de impedancia espectral de las monocapas confluentes con respecto a cultivos no irradiados, indicando una conservación de la integridad de la célula. Es decir, ´estas no presentan muerte por apoptosis ni necrosis lo que implicaría un cambio en la impedancia espectral. En cambio, sí observamos un mayor transitorio en la formación de la monocapa celular de células irradiadas con respecto a células sanas, indicando pérdida de capacidad mitótica ante la radiación.
Resumen en inglés
We have studied the spectral impedance of naked electrodes with respect to the distance between grounded microelectrodes and excited microelectrodes, observing that the resistance and capacitance do not change with the with it. We analyzed the spectral impedance of different microelectrodes, naked and with cells, observing very good repeatability. We developed a new method to predict and estimate the healing radius as a function of time using non-intrusive spectral electrical impedance measurements. We applied the Mesoscopic model on experimental data obtained by ECIS technique during wound and healing assays, being able to estimate the healing radius as a function of time. When comparing the healing radius data obtained by this method with measurements taken from photos at the end of the healing process, a good agreement between them is observed. For these assays we cultured a confluent monolayer of MDCK type II cells on a microelectrode. We then subjected this system to a current high enough to produce severe electroporation in the cells on the surface of the microelectrode, and finally their death. Subsequently, the evolution of the culture was monitored through its spectral electrical impedance. We applied four fractal dimension calculation algorithms on the temporal electrical impedance signal of normal MDCK type II cell cultures monitored by ECIS technique. By these it was shown that the fractal dimension due to micromotion allows to discriminate processes not sensed by the spectral impedance. In this work we subjected cell cultures to ionizing radiation damage, electric current damage and drug exposure to analyze the changes in the fractal structure of the temporal signal. Among the changes presented and detected in the fractal structure is the differentiation between a healthy monolayer and one exposed to a drug, as well as the differentiation between a seeding process and a wound healing process performed by electric current. The four algorithms used were validated by applying them on topological functions of known fractal dimension, a study that determined the necessary conditions for a correct estimation when using experimental data. We developed and tested with known impedances, a novel measurement device using two lock-in amplifiers, to excite a system with two frequencies simultaneously. We then used this configuration to measure spectral impedances of microelectrodes with and without cells. From the electrical impedance measurements we calculated the autocorrelation and cross-correlation for resistance and capacitance signals at low and high frequency excitations. We obtained, at low frequency, correlation times of 20 seconds and evidence of white noise at high frequency. We numerically simulated results of the use of two lock-in’s and the effect of implementing filters of different orders. We performed radiosensitivity assays on normal MDCK type II cultures by monitoring the cell state by ECIS technique. In this work we irradiated cell cultures in confluent state by administering 5, 15 and 17 Gy at different times. We observed no changes in the spectral impedance values of confluent monolayers with respect to nonirradiated cultures, indicating a preservation of cell integrity. That is, they do not show death by apoptosis or necrosis, which would imply a change in spectral impedance. On the other hand, we did observe a greater transient in the formation of the cell monolayer of irradiated cells with respect to healthy cells, indicating a loss of mitotic capacity in the face of radiation.
| Tipo de objeto: | Tesis (Maestría en Ciencias Físicas) |
|---|---|
| Palabras Clave: | Radiosensitivity; Radiosensibilidad; Lock-in amplifiers; Amplificadores lock-in; [Impedance lectrical cell-substrate; Impidencia eléctrica espectral; Signal processing; Procesamiento de señales] |
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| Materias: | Física > Bioimpedancia |
| Divisiones: | Gcia. de área de Energía Nuclear > Gcia. de Ingeniería Nuclear > Cavitación y biotecnología |
| Código ID: | 1150 |
| Depositado Por: | Tamara Cárcamo |
| Depositado En: | 03 Aug 2023 15:44 |
| Última Modificación: | 03 Aug 2023 15:44 |
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