Mitochondria are vital organelles responsible for energy production and they undergo dynamic morphological changes influenced by the cell’s metabolic state. Mitochondrial membrane potential is an essential component in the process of energy storage in the cells. The membrane potential plays a crucial role in the functioning of mitochondria, and it can influence mitochondrial velocity through various mechanisms. Here we report correlation studies on the mitochondrial dynamics and mitochondrial membrane potential in mammalian cells using our home-built sMx-SPIM imaging system. The mitochondria are tracked manually by a self-developed algorithm to find the speed. The speed of mitochondria was determined by targeting mitochondria stained with MitoTracker Deep Red CMXRos dye and measurement of mitochondrial membrane potential was done by tetramethylrhodamine methyl ester (TMRM). Experimental studies were conducted on mammalian cells in alive conditions, and results are provided.
Light sheet fluorescence microscopy (LSFM) works on fluorescence imaging, employing target-specific fluorescent proteins to label regions of interest in a sample. In this way, analyzing and manipulating information from different targets in a specimen requires labeling the specimen using multiple fluorophores. Multispectral imaging helps identify several target molecules simultaneously at once, thereby retrieving information from the entire volume of the sample. Here we report multispectral imaging studies on mammalian cells using our in-house built multispectral LSFM imaging system. Experimental studies were conducted on mammalian cells in alive conditions, and results are provided.
Imaging technology at various scales of spatial resolution is crucial for understanding the physiological and morphological complexity of living organisms. The existing imaging technologies fail to facilitate images of a sample at different magnification levels at a given instant of time. We report the detailed design and instrumentation of an optical imaging system, namely, simultaneous multiple-level magnification selective plane illumination microscopy (with an acronym being given as sMx-SPIM), which addresses one of the technological challenges of imaging biological specimens at different magnification levels simultaneously. The simultaneous magnified views assist in perceiving biological activities occurring over a short period, especially in developmental biology, where the time scales (fraction of seconds) are critical. The proposed imaging system comprises one illumination arm and two detection arms, each of which uses a different magnification to attain multiple magnification levels simultaneously and overcomes the time consumption for changing the objective lens. The system is automated for image acquisition using a custom-built assembly of motion stages and external hardware. Experimental studies are carried out using biological specimens such as Daniorerio and Alliumcepa to validate the home-built sMx-SPIM imaging system at an obtainable spatial (axial) resolution of ∼ 3 to 5 μm.
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