Photon Factory Activity Report 2022 #40 (2023)

AR-NW12A/2022G575

Development of microfluidic devices for in situ diffraction data acquisition
1Swagatha GHOSH1 and Leonard M.G. CHAVAS1,2,*
Department of Applied Physics, Graduate School of Engineering,2
Synchrotron Radiation Research Center,Nagoya University, Furo-Cho, Nagoya, Aichi 464-8603, Japan

1 Introduction
The traditional method for structure determination ofmacromolecular crystals at synchrotrons is to grow crystals
in vitro, then transport them to the synchrotron and collect
X-ray diffraction data. This process is time-consuming andlabor-intensive. Microfluidic devices offer a potential
solution to this problem. Microfluidic devices, such as
small, enclosed chips made with X-ray transparent
membranes/polymers, enable the in situ growth andscreening of protein crystals directly at the synchrotron.
This eliminates the need to transport crystals, saving timeand eliminating the risk of unnecessary and potentiallydamaging crystal handling. In this project, we will develop
microfluidic devices for in situ diffraction data collection.
We will test various formats of microfluidic devices andoptimize the protocols for growing and screening protein
crystals in these devices. We will also investigate the useof microfluidic devices for the structure determination ofprotein-ligand complexes. The development of
microfluidic devices for in situ diffraction data collection
has the potential to revolutionize the field of protein
crystallography. These devices could make structure
determination faster, more efficient, and more accessible toresearchers around the world.
2 Experiments
The protein crystals were cultivated directly inside a
microfluidic chip, utilizing a Lysozyme solution.
Incubation at room temperature for 2 hours yielded
crystals with a maximum dimension of 200 µm.
Optimization of the crystallization conditions ensured thatthe crystal sizes were compatible with both the beam size
and the dimensions of the microfluidic chip channels [1].
One notable advantage of microfluidic chips is their ability
to maintain the crystals in a hydrated environment
throughout the entire course of the experiments. The chip
was securely affixed to a 3D-printed scaffold, which wassubsequently mounted on the goniometer head for precise
sample centering. Crystal positions were registered using
the UGUI user interface, and data collection commenced
directly from the software. To prevent contact between thechip and the beamline hardware, the chip's oscillation
range was restricted to -30º to 30º, resulting in partial data
sets of 60º each.
In situ X-ray diffraction experiments were conducted at theAR-NW12A end station of KEK [2], employing an X-ray
beam with a wavelength of 0.75 Å, focused on the sample
position. The X-ray beam was adjusted to dimensions of200 x 130 µm² to accommodate the crystal size. Fine-slicing data sets were obtained using a Pilatus3 S2Mdetector (Dectris, GmbH), acquiring 6,000 images per data
set with an oscillation range of 0.1º and an exposure timeof 0.1 sec per image.
The integrated software package XDS [3] was employed
for image integration, while the CCP4 software suites [4]were utilized for solving and refining the crystal structures.

Figure 1: Clean diffraction pattern of lysozyme protein
crystals grown in the microfluidic chip. Representative
diffraction pattern obtained from protein crystals growninside the microfluidic chip. The diffraction patterndisplays well-defined Bragg peaks and a minimal
scattering background, indicating the high quality andorder of the crystals and the low-background capacity ofthe microfluidic chip.
3 Results and Discussion
In this study, in situ X-ray diffraction experiments
were performed on protein crystals grown inside a
microfluidic chip. The crystals exhibited diffraction
patterns with excellent quality, allowing for datacollection up to a resolution of 1.8 Å. The diffraction
images showed well-defined Bragg peaks and a highsignal-to-noise ratio, indicating the high quality andorder of the crystals.
Notably, the scattering background originating from themicrofluidic chip was found to be minimal, enabling clear
visualization and accurate measurement of the diffraction

Photon Factory Activity Report 2022 #40 (2023)

intensities. This is particularly advantageous, as a low
scattering background reduces noise and enhances theaccuracy of data analysis.
Furthermore, the handling of the microfluidic chipduring the experiment was straightforward and convenient.
The chip was securely mounted on the goniometer head,
and the oscillation range was carefully controlled toprevent any collisions with the beamline hardware. Thesefactors contributed to the overall ease of use andexperimental efficiency.
Significantly, there were no evident indications ofradiation damage observed during the data collection
process under the aforementioned experimental conditions.
The crystals maintained their diffraction quality
throughout the entire data collection, indicating theirstability and resilience to X-ray exposure. This is asignificant finding, as radiation damage can limit theaccuracy and reliability of structural determination.
Overall, the results demonstrate the successful
application of the microfluidic chip for in situ X-ray(a)diffraction experiments. The high-resolution data obtained,
the minimal scattering background from the chip, the easeof chip handling, and the absence of radiation damage
provide a solid foundation for further structural studies andanalyses. The combination of these favorable features
makes the microfluidic chip a promising tool for future
investigations in the field of structural biology.
Acknowledgment
The authors would like to express their gratitude to thededicated staff members at the AR-NW12A beamline of
KEK for their invaluable assistance and support during theexperimental work. Their expertise and guidance greatly
contributed to the success of this study. The authors would
also like to acknowledge the generous allocation ofbeamtime for this project. This research was conductedunder the beamtime project 2022G575, which provided thenecessary resources and infrastructure for the in situ X-ray
diffraction experiments.

(b)

Figure 2: Microfluidic chip and crystal visualization. (a) 3D-printed scaffold holding the microfluidic chip during insitu X-ray diffraction experiments. The scaffold provides stability and precise positioning of the chip within theexperimental setup. (b) Close-up view of the microfluidic chip revealing the protein crystals grown inside. The chip design
allows for controlled crystal growth and enables direct observation of the crystals during data collection. The positions fordata collection can be saved directly within the UGUI user interface (blue rectangles on the picture).
References[1] R. Vasireddi et al., Micromachines 13, 1365 (2022).
[2] L.M.G. Chavas et al., J. Phys. 425, 012008 (2013).
[3] W. Kabsch, Acta Cryst. D66, 125 (2010).
[4] J. Agirre et al., Acta Cryst. D79, 449 (2023).
* l.chavas@nusr.nagoya-u.ac.jp