Abstract

Intracellular protein concentration is an essential cell characteristic which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on biological cells. Therefore, we have developed a calibration procedure that involves successive imaging of cells in solutions containing different amounts of added protein. This allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/l). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (m3) and 1.0 is an empirical coefficient. We used calibrated TIE imaging to characterize the regulatory volume increase (RVI) in adherent HeLa cells placed in a hyperosmotic solution. We found that while no RVI occurs over the first 30-60 min, the protein concentration fully recovers after 20 h. Furthermore, interpretation of such long experiments may depend on whether protein concentration varies significantly throughout the cell cycle. Our data on HeLa, MDCK and DU145 cells indicate that it remains relatively stable.

Modified Abstract

Intracellular protein concentration is an essential cell characteristic which manifests itself through the refractive index. The latter can be measured from two mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). To improve the accuracy of TIE, we have developed a calibration procedure that allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/l). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (um3) and 1.0 is an empirical coefficient. We used calibrated TIE to characterize variations of C during the cell cycle and its recovery in HeLa cells placed in a hyperosmotic solution. We found that C remains elevated during the first 30-60 min but fully recovers after 20 h.

Research Category

Biology/Ecology

Primary Author's Major

Pre-Medicine/Pre-Osteopathy

Mentor #1 Information

Dr. Michael A. Model

Presentation Format

Poster

Start Date

21-3-2017 1:00 PM

Mudrak.jpg (13222 kB)
Poster

Research Area

Biophysics | Cell Anatomy | Cell Biology | Cellular and Molecular Physiology | Research Methods in Life Sciences

 
Mar 21st, 1:00 PM

Calibrated brightfield-based imaging for measuring intracellular protein concentration

Intracellular protein concentration is an essential cell characteristic which manifests itself through the refractive index. The latter can be measured from two or more mutually defocused brightfield images analyzed using the TIE (transport-of-intensity equation). In practice, however, TIE does not always achieve quantitatively accurate results on biological cells. Therefore, we have developed a calibration procedure that involves successive imaging of cells in solutions containing different amounts of added protein. This allows one to directly relate the output of TIE (T) to intracellular protein concentration C (g/l). The resultant relationship has a simple form: C ≈ 1.0(T/V), where V is the cell volume (m3) and 1.0 is an empirical coefficient. We used calibrated TIE imaging to characterize the regulatory volume increase (RVI) in adherent HeLa cells placed in a hyperosmotic solution. We found that while no RVI occurs over the first 30-60 min, the protein concentration fully recovers after 20 h. Furthermore, interpretation of such long experiments may depend on whether protein concentration varies significantly throughout the cell cycle. Our data on HeLa, MDCK and DU145 cells indicate that it remains relatively stable.