There is an increasing demand for simple and efficient sample delivery technology to match the rapid development of serial crystallography and its wide application in analyzing the structural dynamics of biological macromolecules. Here, a microfluidic rotating-target device is presented, capable of three-degrees-of-freedom motion, including two rotational degrees of freedom and one translational degree of freedom, for sample delivery. Lysozyme crystals were used as a test model with this device to collect serial synchrotron crystallography data and the device was found to be convenient and useful. This device enables in situ diffraction from crystals in a microfluidic channel without the need for crystal harvesting. The circular motion ensures that the delivery speed can be adjusted over a wide range, showing its good compatibility with different light sources. Moreover, the three-degrees-of-freedom motion guarantees the full utilization of crystals. Hence, sample consumption is greatly reduced, and only 0.1 mg of protein is consumed in collecting a complete dataset.

An efficient data collection method is important for microcrystals, because microcrystals are sensitive to radiation damage. Moreover, microcrystals are difficult to harvest and locate owing to refraction effects from the surface of the liquid drop or optically invisible, owing to their small size. Collecting X-ray diffraction data directly from the crystallization devices to completely eliminate the crystal harvesting step is of particular interest. To address these needs, novel microplates combining crystal growth and data collection have been designed for efficient in situ data collection and fully tested at Shanghai Synchrotron Radiation Facility (SSRF) crystallography beamlines. The design of the novel microplates fully adapts the advantage of in situ technology. Thin Kapton membranes were selected to seal the microplate for crystal growth, the crystallization plates can support hanging drop and setting drop vapor diffusion crystallization experiments. Then, the microplate was fixed on a magnetic base and mounted on the goniometer head for in situ data collection. Automatic grid scanning was applied for crystal location with a Blu-Ice data collection system and then in situ data collection was performed. The microcrystals of lysozyme were selected as the testing samples for diffraction data collection using the novel microplates. The results show that this method can achieve comparable data quality to that of the traditional method using the nylon loop. In addition, our method can efficiently and diversely perform data acquisition experiments, and be especially suitable for solving structures of multiple crystals at room temperature or cryogenic temperature.

To resolve photons hungry for weak diffraction samples by the crystallographic method, a double-multilayer monochromator (DMM) was employed on an undulator beamline (BL17UM) at the Shanghai Synchrotron Radiation Facility (SSRF) to provide a focused sub-micron beam with high brightness for macromolecular crystallography experiments. High-quality crystallographic datasets from model protein crystal samples were collected and processed by an existing crystallographic program for structure solution and refinement. The data quality was compared with datasets from a normal silicon crystal monochromator to evaluate the bandwidth of the DMM effect on these crystallographic data. This experiment demonstrates that multilayer optics on an undulator beamline may play a valuable role in satisfying the demands of structure-related research, which requires novel methods.

Serial crystallography is a rapidly advancing experimental technology that has seen significant development in recent years. This technique enables the continuous delivery of a series of protein crystal samples to the X-ray beam, allowing for the collection of diffraction data from a large number of crystals at ambient temperature. Despite its advancements, serial crystallography still possesses considerable potential for further development within synchrotron radiation platforms. Currently, several challenges hinder the progress of this technology, including the preparation of numerous microcrystal samples, methods for sample delivery, data acquisition efficiency, and data processing techniques. The device introduced in this paper is designed to facilitate serial crystallographic experiments at the synchrotron radiation station, employing electrospinning in the vacuum cavity to reduce the average flux, mitigate the effects of air ionization on the Taylor cone, and enhance the stability of Taylor cone during the data acquisition process. The diffraction pattern of lysozyme crystals was successfully acquired with this device at the beamlines of the Shanghai Synchrotron Radiation Facility (SSRF).