Fluorescent Dyes:
Introduction - Quantifying calcium - Definitions&References - Protocols&Examples

Introduction - Choosing a dye - Loading

Fluorescent Dyes - Choosing a dye

There are many commercially available calcium indicators, so it is important to choose the right one for each experiment. Some facts to be considered are listed below:

    • Range of calcium concentration. In general, quantification is possible if calcium concentration is close to Kd value. As a rule of thumb, [Ca2+] has to be between the following values during the experiment:

0,1 · Kd < [Ca2+] < 10 · Kd

    • Instruments. Not only epifluorescence microscopes are suitable for ion imaging, but also confocal microscopes can be used and offer advantages. Illumination source and detection filter/settings have to be suitable for the fluorochrome: the optimum λexcitation for the fluorochrome should be very similar to our excitation light source. Very fast excitation/emission changes are required sometimes to follow fast biological processes.
    • Loading. Many indicators are available as acetoxymethylester (AM), but some of them have to be microinjected or electroporated inside the cell. These compounds require a special equipment and training.
    • Compartmentalization. Some dyes tend to accumulate inside intracellular reservoirs (endoplasmic reticulum, mitochondria, vesicles, etc.). This behaviour depends not only on the dye used, but also on the loading conditions and cell type. There is not a general rule, but sometimes it can be overcome it the loading protocol is modified.
    • Ratiometric/non-ratiometric methods. Though non-ratiometric indicators are easier to measure and quantify, ratiometric indicators are more insensitive to bleaching, concentration differences, optical path, etc. giving robust measurements.
    • Quantum efficiency/photobleaching. A high quantum efficiency gives a stronger fluorescence signal, allowing to work with lower laser intensities. It is important to use low excitation intensities in order to minimize photobleaching. If UV light is used, higher intensities can induce phototoxicity.


Fluorescent Proteins


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