A ratiometric pH reporter for imaging protein-dye conjugates in living cells.

A molecule that transfers energy through bonds from a donor to an acceptor was prepared with a pH sensitive donor function (fluorescein). At pH values above 6.5, minimal energy transfer occurred, and the probe emitted green fluorescence (ca. 520 nm) when excited at the donor (488 nm). Below pH 6.0 however, energy transfer is efficient; hence excitation at the donor causes emission at the acceptor part (600 nm). This probe was used to image a conjugate of the probe with bovine serum albumin that was imported into endosomes or in the cytosol using the noncovalently bound carrier, Pep-1, at 37 and 4 degrees C, respectively. The more acidic environment of the endosomes was conspicuous from the red fluorescence of the probe.