Fluorescence signal calibration. A, pH-calibration
curves. In vitro calibration showing measurements of fluorescence
ratios (for 490- and 450-nm excitations) of 1 μm BCECF in
sodium phosphate buffer at various pH values (dotted lines) and in vivo
calibration showing the same fluorescence ratios measured in G.
margarita germ tubes (solid lines) at varying external pH
values in the presence of 1 μm BCECF-AM, 90
μm potassium chloride, and 4.4 μm
nigericin. Values are means (n = 4–6) (○), with
corresponding se bars. Both calibration curves were
calculated under polynomial fitting (order 4) from measured data
ratios. B, Real-time cell reaction (25 μm from the hyphal tip) of
G. margarita loaded with BCECF to nigericin/potassium
addition after 300 s (vertical arrow) at external pH 5.0. The
fluorescence ratios (right y axis) for 490-/450-nm
excitations and total fluorescence (left y axis) at
450-nm excitation are given. Horizontal arrows indicate the
corresponding axes of the two curves.