When to use the projection assumption and the weak-phase object approximation in phase contrast cryo-EM

Area of Science:

• Cryo-electron microscopy
• Structural biology
• Computational imaging

Background:

• Projection assumption (PA) and weak-phase object approximation (WPOA) are standard for cryo-electron microscopy (cryo-EM) image modeling.
• Advancements in cryo-EM necessitate re-evaluation of these approximations and their limitations for higher resolution.
• Accurate modeling is crucial for interpreting complex macromolecular structures.

Purpose of the Study:

• To critically assess the applicability and limitations of PA and WPOA in cryo-EM.
• To introduce and evaluate the thick-phase grating approximation (TPGA) as a potentially more robust model.
• To provide a practical guideline for selecting the optimal image formation model based on sample properties.

Main Methods:

• Analysis of PA and WPOA under varying conditions to determine their respective applicability.
• Simulation of exit waves using different models, including TPGA, and comparison with multislice calculations.
• Development of criteria (worst-case and average) to define model suitability based on spatial frequency, thickness, and interaction potential.

Main Results:

• Identified specific conditions under which PA and WPOA are valid.
• Demonstrated that TPGA imposes less stringent conditions on interaction potentials and yields comparable results to multislice calculations.
• Quantified the ranges of applicability for PA, PA+WPOA, WPOA, and TPGA.

Conclusions:

• The choice of image formation model significantly impacts cryo-EM simulation accuracy.
• TPGA offers a viable alternative with broader applicability than traditional approximations.
• The developed guideline aids researchers in selecting the most appropriate model for their specific macromolecular complex and experimental parameters.