New Phase Contrast Methods for TEM
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The fundamental limiting factor in biological cryo-electron microscopy (cryo-EM) is the radiation damage of the specimen caused by the electron beam. Due to the limited allowed electron dose TEM images of low-contrast beam-sensitive specimens are characterized by low signal-to-noise ratio (SNR) and rely on phase contrast to extract usable information. The phase contrast method commonly used in cryo-EM is defocus phase contrast (DPC). It utilizes optical aberrations (defocus and spherical aberration) to generate intensity variations in the image in correspondence with phase changes in the specimen wave. DPC is characterized by a sine contrast transfer function (CTF) resulting in a selective contrast where only certain ranges of specimen periodicities are well presented in the image. Of special notice is the low frequency region for which the DPC's performance is particularly poor. This inefficiency is evidenced in DPC images of biological cryo-specimens by their pronounced high-pass filter appearance and low contrast. Overall DPC acts as a band-pass filter and imposes a compromise between image contrast and resolution - increasing the defocus improves the overall contrast but sacrifices resolution, and vice-versa.
The limitations of the defocus phase contrast technique for cryo-EM stimulated in recent years numerous attempts to develop more efficient phase contrast methods. Those are based on devices that modulate the electron wave and are positioned on a diffraction plane inside the microscope. In analogy with light optics such devices are called “phase plates”. The simplest phase plate for TEM is the Zernike thin film phase plate. It consists of a thin material film (~ 25 nm thickness) with a small hole (~ 500 nm) in the centre. Although the simplest, and in part due to its simplicity, the thin film phase plate is the only device that has demonstrated the expected advantages of phase plates in real-world applications.
There are several factors which still prevent the thin film phase plates from being widely utilized. On one side the material and the manufacturing methods for the phase plates are still rather primitive. At present they are prepared by manual transfer of vacuum evaporated carbon films on metal apertures. This introduces a lot of variability in the quality. Carbon films also exhibit “ageing” effects leading to deterioration of the performance of the phase plate with time. Another major reason for the low popularity of the phase plates is the lack of hardware and software support for their efficient and user friendly application.
A part of this project will focus on the investigation of new materials and preparation methods for thin film phase plates in order to improve their performance and quality. In parallel we will work on the development and testing of new hardware and software with the goal of making the phase plate an intrinsic part of the microscope system with seamless integration into existing automated data acquisition routines.
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Dr. Radostin Danev
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