Calibrated brightfield-based imaging for measuring intracellular protein concentration

Area of Science:

• Cell biology
• Biophysics
• Optical imaging

Background:

• Intracellular protein concentration is a key cell characteristic.
• Refractive index is a manifestation of protein concentration.
• Transport-of-intensity equation (TIE) imaging measures refractive index but lacks quantitative accuracy for cells.

Purpose of the Study:

• To develop a calibration procedure for TIE imaging to quantitatively measure intracellular protein concentration.
• To investigate protein concentration changes during regulatory volume increase (RVI) in HeLa cells.
• To assess protein concentration stability across the cell cycle in multiple cell lines.

Main Methods:

• Developed a calibration method using successive imaging in solutions with varying protein content.
• Applied calibrated TIE imaging to adherent HeLa cells undergoing hyperosmotic shock.
• Measured intracellular protein concentration in HeLa, MDCK, and DU145 cell lines over time and across the cell cycle.

Main Results:

• Established a direct relationship between TIE output (T) and protein concentration (C): C ≈ 1.0(T/V).
• Observed no immediate RVI in HeLa cells, but full protein concentration recovery after 20 hours.
• Found that intracellular protein concentration remains relatively stable in HeLa, MDCK, and DU145 cells throughout the cell cycle.

Conclusions:

• Calibrated TIE imaging provides a quantitative method for assessing intracellular protein concentration.
• Cellular protein concentration is stable during the cell cycle and recovers over extended periods during RVI.
• This technique offers a valuable tool for studying cellular biophysical properties.