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Crystallography has made a notable contribution to our knowledge of structural biology. For traditional crystallography experiments, the growth of crystals with large size and high quality is crucial, and it remains one of the bottlenecks. In recent years, the successful application of serial femtosecond crystallography (SFX) provides a new choice when only numerous microcrystals can be obtained. The intense pulsed radiation of X-ray free-electron lasers (XFELs) enables the data collection of small-sized crystals, making the size of crystals no longer a limiting factor. The ultrafast pulses of XFELs can achieve 'diffraction before destruction', which effectively avoids radiation damage and realizes diffraction near physiological temperatures. More recently, the SFX has been expanded to serial crystallography (SX) that can additionally employ synchrotron radiation as the light source. In addition to the traditional ones, these techniques provide complementary opportunities for structural determination. The development of SX experiments strongly relies on the advancement of hardware including the sample delivery system, the X-ray source, and the X-ray detector. Here, in this review, we categorize the existing sample delivery systems, summarize their progress, and propose their future prospectives.

Large gradient high magnetic field (LG-HMF) is a powerful tool to study the effects of altered gravity on organisms. In our study, a platform for the long-term culture of aquatic organisms was designed based on a special superconducting magnet with an LG-HMF, which can provide three apparent gravity levels (µ g, 1 g, and 2 g), along with a control condition on the ground. Planarians, Dugesia japonica, were head-amputated and cultured for 5 days in a platform for head reconstruction. After planarian head regeneration, all samples were taken out from the superconducting magnet for a behavioral test under geomagnetic field and normal gravity conditions. To analyze differences among the four groups, four aspects of the planarians were considered, including head regeneration rate, phototaxis response, locomotor velocity, and righting behavior. Data showed that there was no significant difference in the planarian head regeneration rate under simulated altered gravity. According to statistical analysis of the behavioral test, all of the groups had normal functioning of the phototaxis response, while the planarians that underwent head reconstruction under the microgravity environment had significantly slower locomotor velocity and spent more time in righting behavior. Furthermore, histological staining and immunohistochemistry results helped us reveal that the locomotor system of planarians was affected by the simulated microgravity environment. We further demonstrated that the circular muscle of the planarians was weakened (hematoxylin and eosin staining), and the epithelial cilia of the planarians were reduced (anti-acetylated tubulin staining) under the simulated microgravity environment. Bioelectromagnetics. 2018;39:428-440. © 2018 Wiley Periodicals, Inc.

Varying the temperature has been proven to be beneficial for improving the screening efficiency of protein crystallisation, and thus a crystallisation screening strategy based on this phenomenon can be developed. Such a temperature varying strategy can be applied in practical crystallisation screening, however, there are no guidelines for determining what temperature programme should be utilised. It is therefore necessary to investigate how the temperature programme affects the crystallisation process, so as to help people design a suitable temperature programme. For this purpose, we investigated the effect of temperature programmes on the protein crystallisation (lysozyme, proteinase K, and concanavalin A) that are characterised by different solubility behaviours with respect to temperature. Judging from the reproducibility studies of protein crystallisation with different temperature programmes, we recommend using linear temperature programmes for a moderate time period (24 to 48 h) and a large temperature range according to the properties of the proteins. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

We examined a simple approach, i.e., etching cover glasses using hydrofluoric acid (HF), to determine whether cover glass treatment enhances nucleation in hanging drop protein crystallization. Hen egg white lysozyme and proteinase K were used as the model proteins. We found that the treatment increased the success rate of crystallization. The results indicated that the simple treatment, which is easy to adopt without changing much in the hanging drop method, can be utilized as an alternative method to enhance protein crystallization screens (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The intense inhomogeneous magnetic fields acting on the diamagnetic materials naturally present in cells can generate strong magnetic forces. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can produce three magnetic force fields of -1360, 0, and 1312 T(2)/m, and three corresponding apparent gravity levels, namely 0, 1, and 2-g for diamagnetic materials. In this study, the effects of different magnetic force fields on osteoblast-like cells (MG-63 and MC3T3-E1) viability, microtubule actin crosslinking factor 1 (MACF1) expression and its association with cytoskeleton were investigated. Results showed that cell viability increased to different degrees after exposure to 0 or 1-g conditions for 24 h, but it decreased by about 30% under 2-g conditions compared with control conditions. An increase in MACF1 expression at the RNA or protein level was observed in osteoblast-like cells under the magnetic force field of -1360 T(2)/m (0-g) relative to 1312 T(2)/m (2-g). Under control conditions, anti-MACF1 staining was scattered in the cytoplasm and partially colocalized with actin filaments (AFs) or microtubules (MTs) in the majority of osteoblast-like cells. Under 0-g conditions, MACF1 labeling was concentrated at perinuclear region and colocalization was not apparent. The patterns of anti-MACF1 labeling on MTs varied with MTs' changing under LG-HMF environment. In conclusion, LG-HMF affects osteoblast-like cell viability, MACF1 distribution, expression, and its association with cytoskeleton to some extent.

About 30% of the protein crystals grown in space yield better X-ray diffraction data than the best crystals grown on the earth. The microgravity environments provided by the application of an upward magnetic force constitute excellent candidates for simulating the microgravity conditions in space. Here, we describe a method to control effective gravity and formation of protein crystals in various levels of effective gravity. Since 2002, the stable and long-time durable microgravity generated by a convenient type of superconducting magnet has been available for protein crystal growth. For the first time, protein crystals, orthorhombic lysozyme, were grown at microgravity on the earth, and it was proved that this microgravity improved the crystal quality effectively and reproducibly. The present method always accompanies a strong magnetic field, and the magnetic field itself seems to improve crystal quality. Microgravity is not always effective for improving crystal quality. When we applied this microgravity to the formation of cubic porcine insulin and tetragonal lysozyme crystals, we observed no dependence of effective gravity on crystal quality. Thus, this kind of test will be useful for selecting promising proteins prior to the space experiments. Finally, the microgravity generated by the magnet is compared with that in space, considering the cost, the quality of microgravity, experimental convenience, etc., and the future use of this microgravity for macromolecular crystal growth is discussed.

In this study, effects of diluting either protein or crystallization agents in the droplets on the success rate of protein crystallization was investigated. Diluting the crystallization agent was found to increase the success rate of protein crystallization. Theoretical analysis showed that, concentration ranges of both protein and crystallization agent that can be scanned during the vapor diffusion process are wider with diluting the crystallization agent than that without dilution, resulting in more opportunities for the crystallization solution to be in the nucleation zone. On the other hand, diluting protein could lead to controversial results depending on the location of the initial concentration relative to that of the nucleation zone in the phase diagram. The method of diluting the crystallization agent is therefore proposed as an alternative modification to the conventional vapor diffusion method for obtaining more crystallization conditions in protein crystallization screening. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)