Correlative microscopy approach for biology using X-ray holography, X-ray scanning diffraction and STED microscopy

Area of Science:

• Cellular and Molecular Biology
• Biophysics
• Advanced Microscopy Techniques

Background:

• Correlative microscopy is crucial for integrating structural and functional information in biological samples.
• Existing techniques often require sample manipulation or lack multi-modal integration.
• Visualizing both labeled and unlabeled cellular components simultaneously presents a significant challenge.

Purpose of the Study:

• To develop and demonstrate a correlative microscopy approach integrating holographic X-ray imaging, X-ray scanning diffraction, and STED microscopy.
• To visualize the actin cytoskeleton in heart tissue cells with high resolution and complementary data.
• To establish a framework for interpreting X-ray scattering data in conjunction with fluorescence microscopy.

Main Methods:

• Combined holographic X-ray imaging, X-ray scanning diffraction (scanning SAXS), and stimulated emission depletion (STED) microscopy in a single synchrotron endstation.
• Acquired phase maps from X-ray holography and diffraction patterns from scanning SAXS.
• Mapped fluorescently labeled actin cytoskeleton using STED microscopy.

Main Results:

• Successfully superimposed phase maps from X-ray holography with fluorescently labeled actin cytoskeleton data.
• Observed coincidence between principal directions of anisotropic diffraction patterns and actin fiber directions.
• Identified actin structures within the phase maps generated by holographic recordings.

Conclusions:

• The integrated correlative microscopy approach provides complementary visualization of labeled and unlabeled cellular structures.
• X-ray diffraction patterns offer insights into biomolecular shape and spatial correlations, correlating with actin organization.
• In situ STED recordings can aid in developing models for diffraction data based on co-localization.