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Time-resolved fluorescence microscopy could correct for probe binding while estimating intracellular pH
A Srivastava1, G Krishnamoorthy
1Chemical Physics Group, Tata Institute of Fundamental Research, Mumbai, India.
Analytical Biochemistry
|July 1, 1997
Summary
Accurately measuring intracellular pH with fluorescent probes requires accounting for probe binding to cellular components. This study demonstrates that carboxy SNARF-1 binding can lead to significant overestimation of intracellular pH if not corrected.
Area of Science:
- Cellular Biology
- Biophysics
- Analytical Chemistry
Background:
- Fluorescence ratiometry is a common method for estimating intracellular pH.
- Limitations include variations in probe concentration, light scattering, and photobleaching.
- Probe binding to cellular components is often overlooked.
Purpose of the Study:
- To investigate the extent of carboxy SNARF-1 binding to cellular components.
- To develop a method for correcting probe binding in intracellular pH measurements.
- To validate the developed method using model systems.
Main Methods:
- Time-resolved fluorescence microscopy was employed on various cell types.
- The dual-emission fluorescent pH probe carboxy SNARF-1 was used.
- Distinguishing between free and bound probe populations based on fluorescence lifetime.
Main Results:
- Carboxy SNARF-1 was found to bind significantly to cellular components.
- The fluorescence lifetime of the bound probe (approx. 3 ns) differed from the free probe.
- Intracellular pH estimation requires correction for probe binding to avoid overestimation.
Conclusions:
- Probe binding is a critical factor affecting intracellular pH measurements.
- Time-resolved fluorescence microscopy allows for the resolution of bound and free probe populations.
- Correcting for probe binding is essential for accurate intracellular pH determination, preventing overestimations up to 1 pH unit.