Optimising correlative super resolution and atomic force microscopies for investigating the cellular cytoskeleton

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Correlative imaging enhances cellular ultrastructure analysis by integrating complementary microscopy techniques.
• Super-resolution microscopy and atomic force microscopy (AFM) offer distinct advantages for visualizing cellular components.

Purpose of the Study:

• To develop and optimize sample preparation for correlative super-localized single-molecule microscopy (SMLM) and AFM imaging of the cellular cytoskeleton.
• To investigate the structural organization of microtubules in COS-7 cells using this optimized correlative approach.

Main Methods:

• Optimized sample preparation involving sequential permeabilization and fixation, and controlled Triton X-100 treatment.
• Correlative imaging using SMLM for high-resolution fluorescence localization and AFM for topographical and mechanical property mapping.
• Analysis of microtubule dimensions and organization through integrated SMLM intensity profiles and AFM height measurements.

Main Results:

• Established optimal conditions for sample preparation, preserving microtubule integrity while enabling membrane removal.
• SMLM/AFM revealed distinct information on microtubule widths and cluster arrangements.
• AFM height profiles elucidated the precise ordering of microtubules at intersection points.

Conclusions:

• Correlative SMLM/AFM provides synergistic insights into cytoskeletal architecture, surpassing the information gained from individual techniques.
• The optimized method allows for detailed characterization of microtubule organization, including their orientation and interactions within bundles.