RESUMO
We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation--the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.
Assuntos
Membrana Celular/efeitos da radiação , Fenômenos Fisiológicos Celulares/efeitos da radiação , Campos Eletromagnéticos , Eletroporação/instrumentação , Microscopia de Fluorescência/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Técnicas de Cultura de Células/instrumentação , Eletroporação/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Células Jurkat , Microscopia de Fluorescência/métodos , Doses de Radiação , Integração de SistemasRESUMO
Nanosecond, megavolt-per-meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in the plasma membrane, release intracellular calcium, trigger cardiomyocyte activity, and induce apoptosis in mammalian cancer cells, without the permeabilizing effects associated with longer, lower-field pulses. Dose dependencies with respect to pulse width, amplitude, and repetition rate, and total pulse count are observed for all of these phenomena. Sensitivities vary among cell types; cells of lymphoid origin growing in suspension are more susceptible to nanoelectropulse exposure than solid tumor lines. Simple electrical models of the cell are useful for first-order explanations, but more sophisticated treatments will be required for analysis and prediction at both biomolecular and tissue levels.