The natural occurence of in vivo grown crystals is not very well understood, mostly because of the difficulties to observe such rare events. Using known expression systems, it is now possible to influence this crystal formation within living cells and organisms, which in turns deepen the impact of ivMX on the production of macromolecular crystals directly from their natural environment. Numerous techniques exist for favouring the appearance of in vitro grown crystals, however the current developments are pushing further ivMX as a real alternative for growing macromolecular crystals, not amenable to more classical in vitro crystallisation pipelines.
The state- of -the -art in data collection strategies for macromolecular crystallography involves the collection of serial diffraction data from multiple crystals, and averaging these data in one single set to be as complete as possible. While in the generally accepted serial crystallography approach data are being collected in still images for each crystal, the current approach advocates for collecting data in small wedges rather than still images. Wedges of data have the advantage that they provide with an orientation matrix, in addition of a larger set of images for each crystal, which in turns becomes handy for collecting fully complete and redundant data from fewer crystals.
With the common goal of obtaining unbiased results, data collection strategies are numerous at synchrotron sources, although widely accepted techniques are preferred in the case of macromolecular crystallography experiments. These strategies are generally dictated by the lifetime of the sample, tightly linked to the manner by which it is presented to the x-ray beam. In the current developments, the microfluidic- based sample delivery approach adds one more strategy to the complex data recording of serial crystallography. When designed properly, the microfluidic-based sample sorting and trapping could be adapted at most of the diffraction instruments at synchrotron sources.
Various Serial Crystallography at Synchrotron (SXS) methodologies have been developed and implemented at different x-r-ay sources. Each of these approaches shows advantages over the others, mostly concentrating in rendering the experiments easier for users, as sample preparation in these techniques tend to become different than what it used to be. However, although diffraction SXS experiments proved to be working while rendering spectacular results at x-ray Free-Electron Lasers, all the currently implemented strategies suffer from the high consumption of samples, and as a consequence is sample costly. The main advantage of the currently microfluidic- based positioning of the samples at the x--ray beam interaction point lies in the fact that all the samples are efficiently exposed to the beam. Additionally, using the data collection in wedges of consecutive frames rather than still images, data merging does not rely on Monte Carlo integrations anymore; this strategy of providing with an initial orientation matrix for each sample position helps in speeding data processing, but also in minimizing the amount of necessary crystals to be collected prior to getting a fully complete data set. The amount of necessary sample is therefore strongly decreased, one answer to the concern of users curious in using SXS but reluctant to lose too many of their precious samples.
