Tomographic diffractive microscopy of living cells based on a common-path configuration.

Area of Science:

• Biophotonics
• Cellular Imaging
• Optical Microscopy

Background:

• Accurate refractive-index (RI) mapping is crucial for understanding cellular structures and functions.
• Traditional microscopy methods often require staining, which can affect cell viability and introduce artifacts.
• Existing 3D imaging techniques may suffer from instability and limited resolution.

Purpose of the Study:

• To demonstrate a novel common-path tomographic diffractive microscopy technique for label-free, 3D RI imaging of unstained living cells.
• To compare the performance of the common-path setup against a Mach-Zehnder configuration for improved phase stability and RI resolution.
• To quantify the 3D subcellular RI distributions in live cells.

Main Methods:

• Utilized a common-path off-axis interferometry setup incorporating a diffraction grating to generate a stable reference beam.
• Acquired single-shot phase images at multiple illumination angles.
• Employed optical diffraction tomography principles for 3D RI reconstruction.
• Quantified 3D refractive-index distributions of live HeLa cells.

Main Results:

• The common-path configuration demonstrated significantly lower temporal phase fluctuations compared to a Mach-Zehnder setup.
• Achieved superior refractive-index resolution with the common-path technique.
• Successfully reconstructed and quantified the 3D subcellular RI distributions of live HeLa cells.
• Provided label-free, high-resolution imaging of unstained living cells.

Conclusions:

• Common-path tomographic diffractive microscopy is a robust and high-resolution technique for 3D RI imaging of unstained living cells.
• The developed method offers advantages in stability and resolution over conventional interferometric microscopy.
• This technique enables detailed quantitative analysis of subcellular structures and dynamics in their native state.