Autofluorescence microscopy: a non-destructive tool to monitor mitochondrial toxicity.
ABSTRACT Visualization of NADH by fluorescence microscopy makes it possible to distinguish mitochondria inside living cells, allowing structure analysis of these organelles in a non-invasive way. Mitochondrial morphology is determined by the occurrence of mitochondrial fission and fusion. During normal cell function mitochondria appear as elongated tubular structures. However, cellular malfunction induces mitochondria to fragment into punctiform, vesicular structures. This change in morphology is associated with the generation of reactive oxygen species (ROS) and early apoptosis. The aim of this study is to demonstrate that autofluorescence imaging of mitochondria in living eukaryotic cells provides structural and morphological information that can be used to assess mitochondrial health. We firstly established the illumination conditions that do not affect mitochondrial structure and calculated the maximum safe light dose to which the cells can be exposed. Subsequently, sequential recording of mitochondrial fluorescence was performed and changes in mitochondrial morphology were monitored in a continuous non-destructive way. This approach was then used to assess mitochondrial toxicity induced by potential toxicants exposed to mammalian cells. Both mouse and human cells were used to evaluate mitochondrial toxicity of different compounds with different toxicities. This technique constitutes a novel and promising approach to explore chemical induced toxicity because of its reliability to monitor mitochondrial morphology changes and corresponding toxicity in a non-invasive way.
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ABSTRACT: Abstract We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC(50) values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.Assay and Drug Development Technologies 10/2012; · 1.90 Impact Factor
Article: Mitochondrial network in the heart.[show abstract] [hide abstract]
ABSTRACT: Mitochondria are subcellular organelles that provide energy for the cell. They form a dynamic tubular network and play an important role in maintaining the cell function and integrity. Heart is a powerful organ that supplies the motivation for circulation, thereby requiring large amounts of energy. Thus, the healthiness of cardiomyocytes and mitochondria is necessary for the normal cardiac function. Mitochondria not only lie in the center of the cell apoptotic pathway, but also are the major source of reactive oxygen species (ROS) generation. Mitochondrial morphological change includes fission and fusion that are regulated by a large number of proteins. In this review we discuss the regulators of mitochondrial fission/fusion and their association with cell apoptosis, autophagy and ROS production in the heart.Protein & Cell 06/2012; 3(6):410-8.