Imaging nanometre-scale structure in cells using in situ aberration correction.

Area of Science:

• Cellular and Molecular Biology
• Biophysics
• Optical Microscopy

Background:

• Measuring nanoscale distances within cells is challenging due to light diffraction limits.
• Cells present a complex optical environment that complicates microscopy measurements.
• Existing methods struggle with accurate 3D distance determination at the macromolecular level.

Purpose of the Study:

• To extend high-resolution colocalization for 3D distance measurements of diffraction-limited objects.
• To address and correct for intrinsic chromatic aberration in cellular optical environments.
• To enable nanometer-scale 3D distance analysis within cells using standard widefield fluorescence microscopy.

Main Methods:

• Extension of high-resolution colocalization technique.
• Development of Colocalization and In-situ Correction of Aberration for Distance Analysis (CICADA) method.
• Utilizing multi-color labeled antibodies as intracellular fiducial markers for aberration correction.

Main Results:

• Demonstrated that cells introduce significant and variable 3D chromatic aberration.
• CICADA effectively corrects for wavelength-dependent aberrations in situ.
• Achieved nanometer-scale 3D distance measurements within cells after correction.

Conclusions:

• Standard widefield fluorescence microscopy can achieve nanometer-scale 3D distance measurements with aberration correction.
• The CICADA method provides a robust solution for accurate intracellular distance analysis.
• This technique facilitates deeper investigation into the substructure of macromolecular complexes within living cells.