Single-Cell Sensitive Colorimetric pH Detection Based on Microscope Ratiometric Grayscale.

Area of Science:

• Biomedical Engineering
• Cell Biology
• Analytical Chemistry

Background:

• Accurate intracellular pH measurement is crucial for understanding cellular processes.
• Existing methods can be complex or lack single-cell resolution.
• Developing accessible tools for pH monitoring is essential for biomedical research.

Purpose of the Study:

• To develop and validate a simple, cost-effective method for quantifying intracellular pH at the single-cell level using bright-field microscopy.
• To establish a correlation between color channel ratios and intracellular pH values.
• To demonstrate the method's applicability in monitoring dynamic pH changes in response to stimuli.

Main Methods:

• Utilized a bright-field optical microscope to divide cell images into red (R) and blue (B) color channels.
• Performed a ratiometric operation on the grayscale values of R and B channels to generate ratiometric grayscale images.
• Created a standard curve by incubating HeLa cells at various pH levels (6.00-7.60) with nigericin and K+ buffer.
• Validated the method by measuring intracellular pH in A549 cells and monitoring pH changes in HeLa cells stimulated with hydrogen peroxide.

Main Results:

• Established a strong correlation between pH and the ratiometric grayscale of R and B channels within the pH range of 6.00-7.60.
• Successfully measured the intracellular pH of A549 cells as 7.22 ± 0.01.
• Demonstrated sensitive monitoring of intracellular pH changes in HeLa cells upon hydrogen peroxide stimulation.

Conclusions:

• The developed colorimetric microscopy method provides an easy-to-use approach for accurate intracellular pH detection and quantification at the single-cell level.
• This technique shows significant promise for advancing pH-related single-cell studies in biomedicine.
• The method's simplicity and effectiveness make it a valuable tool for researchers investigating cellular physiology and disease.