Cell imaging

In our laboratory, we predominantly image cells using fluorescence. This is a technique in which cells are labeled with compounds called fluorescent probes or dyes. When excited, these compounds emit light (fluoresce) which we then detect by two different techniques; whole-cell photometry and laser confocal microscopy. The whole-cell photometry technique allows for stable fluorescence recordings from the entire cell over long periods without cell damage. When greater spatial resolution is required, we employ laser confocal microscopy which allows recording of fluorescence from only a specific layer of the cell while excluding regions which are out of focus.

We routinely employ Ca2+-sensitive dyes to examine Ca2+ homeostasis in cardiac muscle cells. In these experiments the dye is loaded into the cytosol, and by recording the fluorescence of the dye we can estimate the cytosolic [Ca2+]. Using the same principles, we employ other dyes to measure cytosolic [Na+] and pH. Fluorescent probes can also be used to examine cell structure. For example, dyes which label the cell membrane or nuclei can be used to determine whether these structures are altered in pathological conditions. With high resolution imaging, the precise localization of specific proteins, such as ion channels and pumps, can also be investigated. In these experiments, the proteins are often labeled with a specific antibody in combination with a secondary antibody which carries the fluorescent probe. By simultaneously labeling two different proteins, we can determine if they are located nearby each other (“co-localized”). We can gain even greater insight into co-localization by employing the newest technique available in our lab, called FRET. This technique relies on measuring fluorescence resulting from energy transfer between two nearby proteins. In combination, these fluorescence techniques give us significant insight into ionic homeostasis, by providing information on both ionic concentrations and the localization of proteins which control these levels.

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