Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.

Area of Science:

• Cell Biology
• Biophysics
• Spectroscopy

Background:

• Microspectrofluorometry allows for the measurement of fluorescence properties in microscopic samples.
• Accurate determination of intracellular parameters like pH is crucial for understanding cell function.
• Digital image processing offers potential for enhanced analysis of microscopic fluorescence data.

Purpose of the Study:

• To develop and validate a digital image processing system for microspectrofluorometry in living cells.
• To accurately map cytoplasmic pH distribution in fibroblasts (3T3 cells).
• To assess the performance and applications of the developed imaging technique.

Main Methods:

• Integration of a microspectrofluorometer with a digital image processing system.
• Utilizing fluorescein-labeled dextran as a pH indicator microinjected into cells.
• Measuring the ratio of fluorescein fluorescence at two excitation wavelengths (489/452 nm) for pH determination.
• Analyzing image processing parameters including linearity, light scattering, signal-to-noise ratio, and spatial resolution.

Main Results:

• The system provides automatically normalized spectroscopic parameters, creating accurate cytoplasmic pH maps.
• The average resting cytoplasmic pH of 3T3 fibroblasts was determined to be 6.83.
• A non-unimodal pH distribution was observed, with a lower mean pH of 6.74.
• Pinosomes showed pH variations depending on their location within the cell.

Conclusions:

• The digital image processing system enables accurate and efficient microspectrofluorometric measurements in living cells.
• The technique provides detailed insights into intracellular pH heterogeneity.
• This ratio imaging approach has broad applicability in cell biology research.