Quantitative reflection contrast microscopy of living cells.

Area of Science:

• Cell biology
• Microscopy
• Biophysics

Background:

• Mammalian cells in culture are crucial models for studying cellular processes.
• Reflection contrast microscopy (RCM) is a technique used to visualize cell-substrate interactions.
• Understanding the optical properties of cellular components can provide insights into their structure and function.

Purpose of the Study:

• To apply reflection contrast microscopy to determine refractive indices within the cortical cytoplasm of various mammalian cell types.
• To differentiate between cellular regions based on their refractive properties and relate these to cell-substrate adhesion.
• To investigate the influence of cell-substrate distance, illumination wavelength, and angle of incidence on reflected light intensity.

Main Methods:

• Utilized reflection contrast microscopy to image mammalian cells (BHK-21, PtK2, Friend, human flia, and glioma cells).
• Acquired images at two different wavelengths (546 and 436 nm) and angles of incidence to analyze interference patterns.
• Calculated refractive indices by solving interference equations, using the glass/medium interface as a reference.

Main Results:

• Refractive indices varied across different cellular regions, ranging from 1.353 to 1.40.
• Higher refractive indices (1.38–1.40) were observed at focal contacts where stress fibers terminate.
• Lower refractive indices (1.354–1.368) were found in areas of close contact and between focal contacts in the cortical cytoplasm.

Conclusions:

• Refractive index mapping using RCM can distinguish between focal and close contacts in mammalian cells.
• The higher refractive index at focal contacts may be attributed to specific cytoskeletal arrangements, possibly a microfilamentous network.
• Intimate cell-substrate attachment is characterized by the absence of an intervening medium layer, influencing optical measurements.