Fig. 4.

Protein-conjugated CypHer5E reports its pH environment. a CypHer5E conjugated to ovalbumin fluoresces upon uptake into intracellular acidic compartments. b Confocal images showing increase in fluorescence 70 min after addition of Ova-Cy to primary hippocampal neurons. c No fluorescence increase is observed in cells pre-treated with NH₄Cl, a weak base that raises the pH of acidic compartments. One 1.0-μm slice is shown. Scale bar is 25 μm. d Quantification of mean fluorescence intensity from cells shown in b and c. Each trace represents an individual cell body either untreated (grey, n = 34) or pre-treated with 10 mM NH₄Cl for 30 min (purple, n = 28). Data pooled from three independent experiments, 3–4 FOV per experiment. Colour bar for look-up table indicates mean fluorescence intensity. e Fluorescence arising from Ova-Cy diluted into buffer of pH 5.4 (purple) or 7.4 (turquoise). Ova-Cy diluted into buffer containing NH₄Cl, pH 7.4 is also shown (orange). The absorbance maximum for CypHer5E at physiological pH 7.4 is 500 nm (left plot) and at pH 5.4 (similar to the pH in a lysosome) is approximately 645 nm. Ova-Cy in acidic (pH 5.4) buffer has increased intensity over neutral pH when excited with 644 nm light. The presence of NH₄Cl has no effect on the spectral properties of the dye