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pH-Sensitive Fluorescent Dyes: Are They Really pH-Sensitive in Cells?
journal contribution
posted on 2013-05-06, 00:00 authored by Xiao-Xiang Zhang, Zhe Wang, Xuyi Yue, Ying Ma, Dale O. Kiesewetter, Xiaoyuan ChenChemically synthesized
near-infrared aza-BODIPY dyes displayed
off–on fluorescence at acidic pH (pKa = 6.2–6.6) through the suppression of the photoinduced electron
transfer and/or internal charge transfer process. The apparent pKas of the dyes were shifted well above physiological
pH in a hydrophobic microenvironment, which led to “turned-on”
fluorescence in micelles and liposomes at neutral and basic pH. Bovine
serum albumin also activated the fluorescence, though to a much lesser
extent. When these small molecular dyes entered cells, instead of
being fluorescent only in acidic organelles, the whole cytoplasm exhibited
fluorescence, with a signal/background ratio as high as ∼10
in no-wash live-cell imaging. The dye 1-labeled cells
remained highly fluorescent even after 3 days. Moreover, slight variations
of the dye structure resulted in significantly different intracellular
fluorescence behaviors, possibly because of their different cellular
uptake and intracellular activation capabilities. After the separation
of cellular components, the fraction of plasma membrane and endoplasmic
reticulum showed the highest fluorescence, further confirming the
fluorescence activation by membrane structures. The fluorescence intensity
of these dyes at different intracellular pHs (6.80 and 8.00) did not
differ significantly, indicating that intracellular pH did not play
a critical role. Altogether, we showed here for the first time that
the fluorescence of pH-sensitive aza-BODIPY dyes was switched intracellularly
not by acidic pH, but by intracellular membranes (and proteins as
well). The excellent membrane permeability, ultrahigh fluorescence
contrast ratio, persistent fluorescent signal, and minimal biological
interference of dye 1 make it an ideal choice for live-cell
imaging and in vivo cell tracking. These findings also imply that
the intracellular fluorescence properties of pH-sensitive dyes should
be carefully examined before they are used as pH indicators.