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

Introduction - Choosing a dye - Loading

Fluorescent Dyes - Introduction

Though there are different dyes used as calcium indicators, BAPTA based compounds are among the most popular for measuring free intracellular Ca2+. These compounds are able to chelate Ca2+ causing a change in their fluorescence spectra.

Fluorescent calcium indicators are normally used to measure intracellular calcium concentration. Only rhod-2 and its derivatives are used to quantify calcium inside an organelle owing to the fact that, in some cell lines, they are sequestered into mitochondria.

Quantification using fluorescent ion indicators can be done using ratiometric or non-ratiometric methods. In general, those compounds that only present an increase in their fluorescence intensity (intensity shift) are used for non-ratiometrics measurements (Figure 1A). A ratio between two fluorescence intensity is calculated in ratiometric measurements: this is performed using ion indicators that, on binding calcium, present an emission spectral shift (Figure 1B) or excitation spectral shift (Figure 1C) or mixtures of indicators. Compounds which have a shift in their spectra on binding to ions are commonly referred as ratiometric indicators.


Intensity shift when bound/unbound to calcium: non-ratiometric measurements



   Free                  Bound


Emission spectral shift: ratiometric measurements



   Free                  Bound


Excitation spectral shift: ratiometric measurements




         Free                           Bound

It is important to consider the advantages and drawbacks of ratiometric and non-ratiometric methods to design a calcium experiment. Other important parameters to evaluate for choosing an indicator are its dissociation constant (Kd) for calcium and/or other ions, excitation and emission wavelength, quantum yield, buffering capacity, loading requirements, etc.

Calcium quantification experiments can be performed in a quantitative or semi-quantitative way. If quantitative experiments are performed, a calibration is recommended.


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