Contrast Enhancement of Long-Range Periodic Structures using Hole-Free Phase Plate

Abstract

Phase contrast transmission electron microscopy is a powerful tool to enhance the image contrast of transparent materials such as ice-embedded biological specimens and polymer materials. In this method, a phase plate, which is placed at the back-focal plane of the objective lens, gives a phase shift for scattered electron waves, resulting in a significant enhancement contrast of specimens in images. Zernike phase plate (ZPP), consisting of a thin carbon film with a small central hole, is first tested practical phase plate [1]. However, ZPP has disadvantages for image quality. Phase contrast of specimens in low spatial frequency does not improve, since the scattered electron passing through a central hole of ZPP does not change their phases. The threshold frequency at the center hole edge is called cut-on frequency. The additional disadvantageous effect of the abrupt cut-on frequency reveals that strong fringes appear.Hole-free phase plate (HFPP) is one of other types of phase plate, consisting only of thin carbon film. In HFPP, the charging on a carbon film due to high-density electron beam (cross-over) acts like a small central hole of ZPP. The size of cross-over on HFPP is smaller than typical size of a central hole of ZPP, which is typically 1 m in diameter. Thus, the cut-on frequency of HFPP is lower than ZPP and phase contrast of larger structures is enhanced by HFPP. Also with HFPP, The fringing effects, which is observed in ZPP, reduces because the width of the fringes is increasing to be wider than sample size with the very small cut-on frequency.In this study, we applied the HFPP to enhance the phase contrast for unstained polymer materials. A 200kV field-emission electron microscope (JEM-2200FS) equipped with Schottky electron source was used to obtain a small cross-over on a HFPP. A HFPP, which is made of 15nm-thick amorphous carbon, is set on the back focal plane of the objective lens of focal length 2.8mm. Figure 1 compares conventional TEM and HFPP-TEM images for 50 nm-thick unstained polymer section (Polystyrene-b-isoprene, Polymer Source Inc.). The conventional TEM images have low contrast, even with the large defocus (Figure 1 (a), (b)). In the HFPP-TEM image, lamellar structures of the polymer are clearly observed (Figure 1 (c)). Figure 2 shows the diffractogram of HFPP-TEM image in Figure 1. Peaks in the diffractogram are corresponding to 0.02 nm -1 in reciprocal space, indicating the lamellar period of specimen is 50 nm (Figure 2). A reason for that such a long periodic structure can be observed by the HFPP-TEM is that the cross-over on a HFPP, which is supposed to be a phase modulation region, is small enough to enhance the low-spatial frequency of the lamellar structures. Figure 3 shows an intensity profile of a cross-over spot, which is formed on a back focal plane and observed in diffraction mode. The measured size of a cross-over on a HFPP in real space (scale in lower 60

Phase plates in the transmission electron microscope: operating principles and applications

“…As compared to alternative PP methods, the salient feature of HFPP is the use of a uniform thin film locally modified by the electron beam itself [1][2][3][4]12,118]. Several microscopic mechanisms can be responsible for HFPP operation (see Section 'Microscopic mechanisms responsible for hole-free phase plate operation') and many different types of HFPP films can be utilized.…”

“…Since under typical conditions, the beam diameter at the HFPP does not exceed a few µm, a 100 µm diameter aperture covered with a HFPP film provides enough film area for hundreds of HFPP experiments. While the steps above are required to retrofit a HFPP in an existing TEM [95,118], instrument manufacturers offer specialized (heated) aperture support and high quality commercial HFPP films [119,120,126]. Commercial HFPP hardware is often equipped with convenient HFPP positioning software and hardware [127][128][129].…”

“…The initial microscope operation is nearly identical to the alignment used for conventional bright field transmission electron microscopy imaging. However, for HFPP imaging, it is necessary to ensure that the BFP cross over coincides with the HFPP film position in the column, as it does for ZPP imaging [1,118,127].…”

“…The main advantages of HFPP compared to other approaches, see Table 3, are the ease of operation and the ability to transfer low (q < 1 nm −1 ) spatial frequencies with minimal contrast ringing when correctly implemented [118]. Objects benefiting from the above advantages include long range magnetic and electric fields, layers inside semiconductor devices and increased visibility of very thin contamination layers.…”