pH-Sensitive Fluorescent Dyes: Are They Really pH-Sensitive in Cells?
2013-05-06T00:00:00Z (GMT) by
Chemically synthesized near-infrared aza-BODIPY dyes displayed off–on fluorescence at acidic pH (p<i>K</i><sub>a</sub> = 6.2–6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent p<i>K</i><sub>a</sub>s 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 <b>1</b>-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 <b>1</b> 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.