Widefield Calcium Imaging with Calcium Indicator Fura2

Burger, Gabriele; Diehl, Simone

January 13, 2013

Widefield Calcium Imaging with Calcium Indicator Fura2

Eukaryotic cells Widefield_Application_Letter_Fura_01_Sep07_en-1.jpg

In eukaryotic cells Ca²⁺ is one of the most widespread second messengers used in signal transduction pathways. Intracellular levels of Ca²⁺ are usually kept low, as Ca²⁺ often forms insoluble complexes with phosphorylated and carboxylated compounds. Typically cytosolic Ca²⁺ concentrations are in the range of 100 nM. In response to stimuli Ca²⁺ may either be released from external medium or internal stores to raise the Ca²⁺ concentration.

As Ca²⁺ cannot be visualized directly in living cells, it may be imaged indirectly by fluorescent Ca²⁺ indicators.

Ca²⁺ dyes

Ion sensitive dyes are fluorescent molecules which reversibly bind to specific ions. These dyes are sensitive to changes in ion concentration either by increase or decrease of fluorescence. A measure for the affinity of ion binding to the dye is the dissociation constant (Kd). The lower the Ca²⁺ binding affinity the higher the Kd (Ca²⁺). There are two major groups of ion-sensitive dyes: 1. Single wavelength dyes, like Fluo3 and Calcium Green which are suitable for non-ratiometric measurements. The intensity of the emitted fluorescence light increases proportional to free ion concentration. 2. Dual wavelength dyes are used for ratiometric measurements. These indicators can also be divided in two groups:

Dual emission ion indicators:
These dyes are excited at one wavelength and show a shift in their emission spectra upon binding specific ions. A well-known and often applied indicator of this group is Indo1.

Dual excitation ion indicators:
These dyes are excited at two different wavelengths The intensity of the emitted fluorescence light changes depending on the ion concentration. Fura2 is one of the most frequently used ratiometric Ca²⁺ indicator.

Fura2

The Ca²⁺ unbound form of Fura2 gets excited at 380 nm and the Ca²⁺ bound form at 340 nm. The emitted light is measured at around 510 nm. The fluorescence intensity increases at 340 nm with increasing Ca²⁺ concentration and decreases at 387 nm for the unbound form. There are some techniques to introduce the fluorescent Ca²⁺ indicator into cells, like ester loading, microinjection, chemical loading techniques or diffusion from patch-clamp pipettes.

For further information on different Ca²⁺ indicators, handling of the dye, labeling of the cells and cell loading, please refer to the Molecular Probes Handbook and Takahashi et al. (see References).

Fig. 1: Fluorescence excitation spectra of Fura2 at different calcium concentrations. Reference: Molecular Probes.

Benefit of ratiometric measurement
Artifacts due to uneven dye distribution, leakage of dye, photo bleaching and unequal cell thickness may be avoided by ratioing methods. In a typical experiment where you detect Ca²⁺ changes over time a calibration must be done. Calibration is possible in vitro or in vivo. For in vivo calibration the ratio of maximum and minimum Ca²⁺ concentrations is measured. These values may be obtained within the same experiment. For the maximum amount of bound Ca²⁺ indicator you may add an ionophore like Ionomycin or Bromo-A2318 to equilibrate the Ca²⁺ concentration. To get the minimum Ca²⁺ concentration you may apply an EGTA buffered solution to chelate all Ca²⁺.

Leica systems configuration

The system must be equipped with external filter wheels and EL6000 light source. The external filter wheels contain special excitation filters for Fura2 at 340 nm and 387 nm. Additionally you need the according filter cube consisting of a dichroic beamsplitter and an emission band pass filter for selection of the wavelength with the maximum at 510 nm.

References

Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260 (1985) 3440–3450.
Takahashi A, Camacho P, Lechleiter JD, Herman B: Measurement of Intracellular Calcium. Physiological Rev 79 (1999) 1089.