Generalization of the Ratiometric Method to Extend pH Range Measurements of the BCECF Probe.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Cell Biology

Background:

• Fluorescent probes are essential for biological pH measurements.
• Existing probes fall into two groups: single emission peak or dual emission peak.
• Ratiometric measurements enhance accuracy by normalizing probe concentration effects.

Purpose of the Study:

• To develop and validate a methodology for evaluating the precision of ratiometric pH measurements.
• To determine optimal excitation wavelength combinations for pH determination in biological samples.
• To apply this methodology to measure pH drift in cell culture media.

Main Methods:

• Classification of fluorescent pH probes based on emission characteristics.
• Implementation of ratiometric pH measurement techniques using dual excitation wavelengths.
• Development of a methodology to assess measurement precision using multiple excitation wavelengths.
• Application of the BCECF probe for pH drift analysis in cell culture.

Main Results:

• The proposed methodology precisely evaluates ratiometric pH measurements.
• Optimal wavelength combinations were identified for accurate pH determination.
• The method successfully measured pH drift in cell culture medium.
• The technique demonstrated high precision and an extended pH measurement range (acidic to basic).

Conclusions:

• The developed methodology offers a precise approach for ratiometric pH determination in biological systems.
• This technique enhances the reliability and range of fluorescent pH measurements.
• It is particularly useful for monitoring dynamic pH changes in environments like cell culture media.