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Measuring Phagosome pH by Ratiometric Fluorescence Microscopy
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pH-sensitive fluorescent dyes: are they really pH-sensitive in cells?

Xiao-Xiang Zhang1, Zhe Wang, Xuyi Yue

  • 1Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States.

Molecular Pharmaceutics
|March 8, 2013
PubMed
Summary

New near-infrared aza-BODIPY dyes show "on" fluorescence in cell cytoplasm, not just acidic parts. This is due to activation by intracellular membranes, making them ideal for long-term live-cell imaging.

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Area of Science:

  • Chemical Synthesis and Photophysics
  • Cellular Imaging and Biology

Background:

  • Near-infrared (NIR) aza-BODIPY dyes are synthesized for potential bioimaging applications.
  • pH-sensitive dyes often rely on acidic environments for fluorescence activation, limiting their utility in complex biological systems.

Purpose of the Study:

  • To investigate the intracellular fluorescence behavior of novel NIR aza-BODIPY dyes.
  • To determine the factors responsible for fluorescence activation within live cells.

Main Methods:

  • Synthesis of NIR aza-BODIPY dyes with varying structures.
  • In vitro studies using micelles, liposomes, and bovine serum albumin to assess fluorescence response.
  • Live-cell imaging experiments to evaluate intracellular localization, fluorescence intensity, and signal persistence.
  • Cellular component fractionation to identify fluorescently labeled structures.

Main Results:

  • Dyes exhibited pH-dependent off-on fluorescence (pKa = 6.2-6.6) in solution, with fluorescence activated in hydrophobic environments (micelles, liposomes) at neutral/basic pH.
  • Intracellularly, dyes showed widespread cytoplasmic fluorescence rather than localization solely in acidic organelles.
  • High signal/background ratios (∼10) and persistent fluorescence (up to 3 days) were observed in live-cell imaging.
  • Plasma membrane and endoplasmic reticulum fractions showed the highest fluorescence, indicating membrane-mediated activation.
  • Intracellular pH variations (6.80-8.00) did not significantly affect fluorescence intensity.

Conclusions:

  • Intracellular fluorescence of these aza-BODIPY dyes is primarily activated by intracellular membranes and proteins, not acidic pH.
  • Dye 1 demonstrates excellent membrane permeability, high fluorescence contrast, and long-term stability, suitable for live-cell imaging and in vivo tracking.
  • The findings highlight the need to carefully evaluate intracellular activation mechanisms of pH-sensitive dyes before their application as pH indicators.