Methodological aspects of pressure loading of Fura-2 into Characean cells

Abstract

Four different fura-2 compounds were tested for the application in Characean cells (fura-AM; fura-C₁₈; fura- K₅; fura-dextran; MW = 10 kDa). It is demonstrated that Characean cells impose special problems when cytosolic pCa has to be measured with fluorescent ratio dyes. Fluorescence (λ_ex=340 nm) from the dye which had diffused from the cytosol to the huge central vacuole with milimolar Ca²⁺ concentrations overrides the signal from the cytosol and makes Ca²⁺ -quantification difficult. This can be avoided by pressure injection of fura-dextran. Because of inhomogeneities in dye concentration or in thickness of the cytoplasmic layer, cytoplasmic streaming causes high noise or pretend oscillations in pCa if data are obtained by subsequent image grabbing. In addition, vesicles filled with high 'concentrations of dye may sometimes be expelled into the vacuole during the loading procedure enhance this effect. These sources of inhomogeneities can be minimized by loading fura-dextran via the neighbouring cell. The slow loading procedure through the plasmodesmata takes 1–10 h. It results in a more homogeneous distribution of the dye. The operation of the new method is illustrated by the measurement of Ca²⁺ -transients during action potentials, the temperature dependence of the fluorescence signal in vivo and in vitro and the butyrate-induced elevation of [Ca²⁺]_c. Fura-AM was found not to be well suited for use in algal cells. Fura-C₁₈ has toxic effects and induces clotting of the cytoplasm. In addition, some aspects of the properties of dextran-derivates are discussed.