CALCIUM IMAGING BY DIGITAL FLUORESCENCE MICROSCOPY.

Since the introduction of synthetic calcium dyes, fluorescence microscopy has enabled imaging of intracellular Ca²⁺ in single cells. The most popular probe is fura-2, a ratiometric dye that has considerable advantages over previous probes. The cells are loaded with fura2/AM, a permeable derivative that is cleaved by intracellular esterases releasing fura2 acid in the cytosol where it reports changes in Ca²⁺ concentration. For fura2 measurements, cells are plated at about 0.5x10⁶ cells/ml on 12 mm glass coverslips treated with poly-L-Lysine and loaded with 4µM fura2/AM or fura4F/AM for 60 min at room temperature in standard medium containing (in mM) NaCl, 145; KCl, 5; MgCl₂, 1; CaCl₂; glucose, 10; HEPES/NaOH, 10 (pH: 7.42) and placed on the stage of an inverted microscope (Nikon Diaphot) maintained at 37 ºC. Then they are perfused with prewarmed medium and epi-illuminated alternately at 340 and 380 nm. Light emitted above 520 nm is recorded with a Magical Image Processor (Applied Imaging). Pixel by pixel ratios of consecutive frames were captured and [Ca²⁺]_cyt is estimated from these ratios. New, improved fluorescent Ca²⁺ probes can be found in the Invitrogene website.

For further details see:

Redistribution of Ca2+ among cytosol and organella during stimulation of bovine chromaffin cells.   Villalobos C, Núñez L, Montero M, García AG, Alonso MT, Chamero P, Alvarez J,  García-Sancho J (2002). FASEB J  16, 343-353.

Cell proliferation depends on mitochondrial Ca2+ uptake: inhibition by salicylate.   Núñez L, Valero RA, Senovilla L, Sanz-Blasco S, García-Sancho J, Villalobos C (2006). J Physiol 571, 57-73.

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