**1. Introduction**

In recent years, in order to efficiently obtain the structure of protein, various processing steps of the protein crystallography have been improved and optimized, especially with the development of in situ X-ray crystallography [1]. The method of in situ diffraction has been developed to directly collect datasets from the location where crystals were grown using X-ray diffraction, which eliminates the process of transferring the crystal sample to nylon loop and avoids the influence of human factors on the quality of the crystals. This method was originally used to screen crystals and verify the quality of crystals, but now this method is mainly used to collect multiple data sets to obtain high-resolution protein structures, because in situ diffraction can greatly improve the efficiency of data collection, and at the same time, is very suitable for some special crystals. For example, some proteins can only produce microcrystals, and microcrystals are fragile to transfer with a normal nylon loop. In addition

to the development of in situ data collection methods, microcrystals are sensitive to radiation damage, so it is necessary to collect data from multiple crystals in a small wedge and integrate the diffraction images of multiple crystals into a full dataset. Since the determination of radiation-sensitive crystal structures requires a large number of crystals, high-throughput data collection methods are also needed to improve efficiency. A number of successful devices have already been designed for in situ data collection at different light sources. So far, microfluidic devices [2–4], chips [5], and regular 96-well crystallization plates [6] are the main methods reported for in situ data collection.

Microfluidic devices including capillaries and nano-droplets are usually used for protein crystallization and then directly for in situ data collection at room temperature using such devices. Microfluidic devices provide sufficient convectionless space for high-throughput crystal growth and screening. For example, Pinker et al. reported a microfluidic device ChipX [7] that can, not only obtain high-quality crystal and diffraction data, but also perform in situ characterization without directly processing crystals. Furthermore, the microfluidic device ChipX is good for in situ data collection for fragile crystals. However, ChipX is not suitable for flash-cryocooling of crystals, which will cause the liquid to freeze. In addition, the microfluidic device based on capillary is also used for in situ data collection of protein crystals [8]. It has been reported that two sets of devices containing aqueous solution and oil respectively are designed to cooperate with the capillary [9]. The aqueous solution including the protein solution and precipitant is used for protein crystallization, and the oil is used to separate the aqueous solution to form a separate crystallization environment, and then the microcapillary containing the crystal is directly installed on the beamline station for data collection after the crystallization experiment. The in situ method based on nano-droplets is also generally implemented by the microfluidic device [10]. The crystals in the capillary gradually move to the surface of the nano-droplets and are fixed near the droplet interface. During the whole process, there is no special operation to fix the crystals, crystals are fixed by the high surface tension of the droplet and used for further data collection. Furthermore, the in situ method based on nano-droplets can realize free interface diffusion crystallization and large-scale preparation of monodisperse crystals, which avoids crystal accumulation. However, the data is collected in the liquid phase at room temperature so that the crystal is susceptible to radiation damage [11].

Chip and film were reported to transfer or grow crystals, and then mount them on the goniometer head for data collection. These methods do not limit the size of crystals. It has a high hitting rate of X-ray during data collection. The amount of protein used is small and only a few hundred crystals are needed to obtain a full dataset of protein structures [12–15]. The chip material generally uses quartz with high light transmittance and low background scattering [16]. X-rays have a high hitting rate to the crystal using a chip-based sample delivery method. Using micro-nano processing technology, holes or grooves are etched on the chip, and then the crystal is fixed in the holes and grooves on the chip for diffraction data collection. Zarrine-Afsar et al. first proposed the application of the chip-based sample-delivery method for protein crystal data collection [17]. They grew the crystals in situ on a chip with a polyimide film attached to the lower end. The chip has a grooved array (silicon mesh). By adding glass beads in the grooved area of the chip, the random orientation of the crystals is induced by increasing the roughness. The chip can be used for the in situ growth of macromolecular crystals and serial data collection. When data is collected, it is allowed to rotate at a certain angle, and collect multiple sets of diffraction data for one crystal. The sample consumption is small and it is increased by adding glass beads. The surface roughness makes the crystals oriented randomly. Some structures have been obtained by this method. However, there are chip grids, polyimide films, and crystalline solutions that will cause high background scattering. A thin film, transferred with crystals, fixed to the goniometer head for diffraction data collection was reported by Li et al. [18]. Li et al. designed a bracket, and then attached the polyimide membrane to the resin bracket with SuperGlue. The microcrystals were transferred from the coverslip to the surface of the polyimide membrane by a micromesh loop, a glass capillary with a tip diameter less than 100 μm was used to remove excess liquid, and then crystals were sealed with another polyimide film to protect it from dehydration, but human factors

are added during the sample transfer. Baxter et al. designed a high-density grid [19], covered with a polymer film or sleeve, for efficient data collection for multiple crystal samples, incubation chambers have been developed to support crystallization experiments on grids, but the grid cannot screen the crystallization conditions because there is only one type of desiccant in the incubation chamber.

Currently, a particular research interest is the possibility of data collection directly from crystallization plates, normal size 96-well plates are reported for crystal screening and in situ data collection [20]. With the development of various systems for plate setup and handling, the crystallization plates have been standardized to achieve compatibility with some beamline platforms, for example, the Structural Biology Center (SBC) beamline 19-ID, located at the Advanced Photon Source, USA. Significant progress has been made to provide plates with a low X-ray absorption profile and scattering properties, such as the MiTeGen In situ–1 Plate (MiTeGen, Inc.). However, with these kinds of methods, it is difficult to focus and align the crystals to the beam position once the plates rotate to a small wedge angle.

Here, a set of simple and inexpensive microplates (plate A and B) is designed for screening crystallization conditions and in situ data collection at room temperature or cryogenic temperature. Plate A is used for setting drop vapor diffusion crystallization. The assembly of plate B and a specially designed crystallization plate is applied for the hanging-drop vapor-diffusion method. The 12.5 μm thick Kapton membranes are used for crystal growth and sealing up the microplates. Lysozyme is used as the model protein for crystal growth, and lysozyme crystals are used to verify the practicability of the new device and method. Structure obtained by in situ method is compared with that obtained by the traditional method. The grid scanning, implemented from Blu-Ice [21] was used at the Shanghai Synchrotron Radiation Facility (SSRF) BL18U1 beamline for sample location and data collection. Results show that the devices can be used for screening crystallization conditions and in situ data collection from multiple crystals.
