New Phase Contrast Methods for TEM
The fundamental limiting factor in biological cryo-electron microscopy (cryo-EM) is radiation damage of the sample caused by the electron beam. This limits the allowed electron dose and leads to low signal-to-noise ratio (SNR) in the images which necessitates the use of phase contrast to gather usable information. The phase contrast method commonly used in cryo-EM is defocus phase contrast (DPC). It relies on optical aberrations (defocus and spherical aberration) to generate intensity variations in the image in correspondence to phase changes in the electron wave caused by the specimen. DPC is characterized by a sine contrast transfer function (CTF) resulting in a selective contrast enhancement where only certain ranges of specimen periodicities are represented in the image. Of special notice is the low frequency region for which the DPC's performance is particularly poor. This inefficiency is evidenced by the low overall contrast and high-pass filter like appearance of DPC images. Overall, DPC imposes a compromise between image contrast and resolution - increasing the defocus improves the overall contrast but sacrifices resolution, and vice-versa.
In recent years the limitations of defocus phase contrast cryo-EM stimulated attempts to develop more efficient phase contrast methods. Those are based on devices that modulate the electron wave and are typically situated on a diffraction plane inside the microscope. In analogy with light optics such devices are called “phase plates”. The oldest phase plate design for TEM is the Zernike thin film phase plate (ZPP). It consists of a thin (~ 25 nm) material film with a small (~ 500 nm) hole in the center. It was the first phase plate to be used in cryo-EM applications and demonstrate the usefulness of phase plates. Despite the encouraging initial results the ZPP plate had several practical disadvantages which prevented its wide adoption. The ZPP has a very short usable life of about a week after which it has to be replaced. It requires precise centering of the electron beam in the small central hole which makes automated data acquisition practically impossible. Last but not least, it produces fringe artefacts in the images due to the sudden onset of the phase contrast CTF at the edge of the central hole.
As a successor to the Zernike phase plate we recently developed the Volta phase plate (VPP) which has similar but simplified design. It comprises a thin (~ 10 nm) continuous amorphous carbon film constantly heated to ~ 250oC. Unlike the Zernike phase plate the VPP does not have a central hole. The phase shift is created “on-the-fly” by the interaction of the central diffraction beam with the film. This characteristic makes the VPP very convenient to use because one does not have to precisely center it but just pre-irradiate an arbitrary position on the film. Consequently, a large open area of the film can be used to create multiple phase plates and thus the VPP is easy to adopt in automated data acquisition routines. The VPP has a virtually unlimited service lifespan. In our lab we have used some of the VPPs for more than two years. Unlike the Zernike phase plate the VPP does not produce fringe artifacts in the images which gives them a more “natural” look.
At the moment our research is focused on cryo-EM applications of the Volta phase plate and further optimization of the experimental protocols. We are applying the VPP with the two major 3D structural investigation cryo-EM techniques: single particle analysis and cryo-tomography. The results show big improvements in contrast, visibility of fine specimen features and capability to solve the structures of small proteins.
EP2797100A1, US9129774B2, Bart Buijsse, Radostin Danev, “Method of using a phase plate in a transmission electron microscope”, 2015/09/08.
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