Accurate, automatic determination of astigmatism and phase with Ctfplotter in IMOD

Area of Science:

• Microscopy
• Image Analysis
• Computational Biology

Background:

• Accurate determination of Contrast Transfer Function (CTF) parameters, including defocus and astigmatism, is crucial for high-resolution cryo-electron microscopy (cryo-EM).
• Existing methods for CTF estimation, such as Ctffind, Gctf, and Warp, primarily rely on fitting parameters to two-dimensional power spectra, which can be less reliable at higher tilt angles.

Purpose of the Study:

• To introduce Ctfplotter, a novel program within the IMOD software package, designed for flexible and reliable determination of CTF parameters in tilt series images.
• To evaluate the performance of Ctfplotter in estimating astigmatism and defocus compared to existing software.

Main Methods:

• Ctfplotter employs a novel approach by measuring defocus in one-dimensional power spectra, rotationally averaged over restricted angular ranges, to determine astigmatism.
• The program incorporates features for optimizing power spectrum sampling and determining angle-dependent fitting ranges.
• Results can be aggregated over multiple tilt angles (typically 5) to further reduce astigmatism estimation variability.

Main Results:

• Ctfplotter demonstrates more reliable astigmatism estimation compared to Ctffind, Gctf, and Warp, particularly at higher tilt angles.
• The method provides improved defocus and phase estimates.
• Integration with SerialEM software enables real-time focus and astigmatism determination during image acquisition.

Conclusions:

• Ctfplotter offers a robust and flexible solution for CTF parameter determination in cryo-EM tilt series.
• Its novel approach enhances the accuracy of astigmatism and defocus measurements, contributing to improved image quality and resolution in cryo-EM.
• The program's automated features and integration with acquisition software facilitate its use in various applications.