With recent advances in sample preparation and TEM instrumentation, cryo transmission electron microscopy (cryo-TEM) has become an important and powerful technique in structural biology to analyze specimen in their native state [1]. These frozen, hydrated specimen however show only weak image contrast. Nearly all image information is contained in a phase change of the scattered electron beam. This phase information can be transferred to image contrast by influencing the contrast transfer function (CTF) of the TEM. This is commonly done by changing the focus ("defocusing"). Unfortunately, at the same time, image resolution is lowered. In addition, cryo-samples are too beamsensitive to record multiple images of the same spot to compensate for the worsened resolution caused by the defocusing.Using a phase plate instead of defocusing is a viable alternative to gain contrast and preserve resolution. The phase plate is inserted into the back focal plane of the TEM at or near Gaussian focus, modulating the CTF. The phase of the scattered electrons is shifted by π/2. This allows maximum contrast transfer. Thus, phase plates can show structures that are not visible in the original data gained by defocusing. This holds particular advantage for objects which cannot be averaged [2].So far, the main obstacles for the widespread use of phase plates were their limited lifetime and the prevalence of image artifacts. Here, we present procedures and techniques that solve the above mentioned problems.As an alternative to prevalent carbon-film and multi-layer Zernike type phase plates, we developed a new, self-supporting, 1-layer thin film metal phase plate. The phase shift of π /2 is controlled by the thickness of the layer with respect to the desired accelerating voltage. To let pass the unscattered electron beam, a central hole is milled into the film using a focused ion beam system (FIB).Furthermore, we devised an in-situ conditioning process that removes any remaining hydrocarbons from the phase plate. After this treatment, the phase plate shows no signs of ageing.In addition, we implemented the rocking beam technique to remove eventual charging of the phase plate aperture [3] and to realize a time-averaged image. Alternatively, we let rotate the phase plate aperture with similar results [4,5,6]. The effects of the "rocking" technique are clearly shown in figure 1.With these most challenging obstacles removed, automation and system integration will be the last tasks to be solved before phase plates will become a widespread feature in biological TEM.
This paper demonstrates why VCSEL-based transmitter systems are not able to capitalize on higher-order PAM modulation formats (such as PAM-4) to push the link data rate towards 100 Gbit/s with BER of better than 10−5. First, the non-linear and biasing-dependent VCSEL behavior is analyzed in great detail to prove why state-of-the-art VCSELs with linear equalization are not able to reach these bandwidth targets. Consequently, based on an enhanced VCSEL model, a digital non-linear transmit equalizer is proposed to overcome voltage-dependent relaxation frequency shifts as well as data-dependent VCSEL bandwidth variations. The proposed digital non-linear equalizer is compared with a standard linear FFE structure using PAM-4 transmission experiments of 100 Gbit/s and beyond. This way, the proposed non-linear equalizer reveals a BER improvement of more than 30-fold compared to a linear FFE at a data rate of 80 Gbit/s. Finally, at 112 Gbit/s PAM-4 the non-linear equalizer was successfully demonstrated at a BER of 1.5·10−7.
Single mode (SM) VCSELs, produced in industrial 4” technology, are suitable for 100Gb/s PAM2 and >160Gb/s PAM4 data transmission. >107Gb/s transmission over 1km of multimode (MM) fiber at 850nm and 910nm is realized.
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