February 2025 was an intense and exciting month for Nogly Lab. Within just two weeks, our team carried out back-to-back time-resolved serial femtosecond crystallography (TR-SFX) experiments at two of the world’s leading X-ray free-electron laser facilities: the European XFEL in Schenefeld, Germany, and SwissFEL at the Paul Scherrer Institute in Villigen, Switzerland. The goal of both experiments was to capture the ultrafast structural dynamics of the light-sensitive LOV (Light–Oxygen–Voltage) domain from Chlamydomonas reinhardtii (CrLOV) — a protein at the heart of our research into optogenetic photoreceptors.
Growing crystals for serial crystallography
Every successful XFEL experiment starts weeks — sometimes months — before the beamtime itself. Unlike conventional crystallography, where a single large crystal is carefully mounted and rotated in the X-ray beam, SFX requires thousands to millions of tiny microcrystals, each exposed to just a single femtosecond X-ray pulse before it is destroyed. The crystal must diffract before it is obliterated — the famous “diffract-before-destroy” principle that makes XFELs uniquely powerful for radiation-sensitive and time-resolved structural studies.
Left: Initial CrLOV crystals grown in batch crystallization. Right: After optimizing conditions — uniform and densely packed microcrystals ready for the XFEL beam.
For our CrLOV experiments, the crystallization team in Kraków spent weeks optimizing batch crystallization protocols to produce microcrystals of the right size and density. Serial crystallography at an XFEL demands not only the correct crystal dimensions — typically a few to tens of micrometres — but also homogeneity, high crystal density, and stable suspensions that can survive transport across Europe.
Why do the crystals look so tiny? Because they are! Serial crystallography relies on microcrystals — here viewed under a microscope and displayed on a monitor — each one just a few micrometres across.
European XFEL — SPB/SFX beamline (7–10 February 2025)
Our first stop was the European XFEL in the Hamburg metropolitan area — the world’s first high-repetition-rate hard X-ray free-electron laser. The European XFEL produces extremely bright and ultrashort X-ray flashes at a unique burst-mode structure, delivering up to 27,000 pulses per second. We conducted our experiment at the SPB/SFX (Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography) instrument, one of the flagship beamlines for structural biology, designed for structure determination at atomic or near-atomic resolution with a strong emphasis on time-resolved dynamics from femtoseconds to milliseconds.
The Kraków team at the European XFEL, ready for four days of round-the-clock data collection.
A dense CrLOV microcrystal slurry packed into a sample tube. The characteristic yellow colour comes from the FMN chromophore bound within the LOV domain.
At SPB/SFX, our sample was delivered to the X-ray beam via a Gas Dynamic Virtual Nozzle (GDVN) — a 3D-printed two-channel device that uses a coaxial helium gas stream to focus a liquid jet containing the crystal suspension down to just a few micrometres in diameter. The GDVN produces stable, fast jets at remarkably low flow rates — on the order of tens of microlitres per minute — critical for precious protein samples. Jet velocities can exceed 25 m/s, fast enough to replenish crystals between the megahertz X-ray pulses, where diffraction patterns are recorded by the AGIPD detector at up to 3,520 images per second.
The team together with a part of collaborators at the SPB/SFX beamline of the European XFEL. Beamtime is a collaborative effort — our group worked alongside beamline scientists around the clock.
The four days at European XFEL were as demanding as they were rewarding. XFEL experiments run non-stop — shifts rotate through morning and afternoon, and every hour counts. The intensity of the work is matched only by the thrill of watching real-time diffraction data appear on the monitoring screens.
Left: This happiness when good data is finally being collected lifts team spirits up. Right: An unexpected work of art — used-up sample forms a delicate “Christmas tree” inside the GDVN catcher, providing an unexpected joy.
SwissFEL — Cristallina beamline (21–24 February 2025)
Just ten days after returning from Hamburg, our team was on the road again — this time heading south to the Paul Scherrer Institute in Switzerland. We carried the same CrLOV sample, now adapted for an entirely different sample delivery approach.
At SwissFEL, we worked at the Cristallina experimental station — the third endstation of the ARAMIS hard X-ray beamline, in user operation for SFX since January 2024. Our team has a deep connection to this beamline — we contributed LOV1 crystals to commission the SwissMX endstation for pump–probe experiments, resulting in an important publication on eliminating light contamination in fixed-target TR-SFX (Gotthard, Flores-Ibarra et al., IUCrJ 2024). You can read more in our earlier news post and on our Research page, where we describe the LOV domain photoactivation results in detail.
Unlike the liquid-jet approach at European XFEL, our Cristallina experiment employed the SwissMX fixed-target system with PSI-developed MISP (Micro-Structured Polymer) chips. These precision-fabricated polymer supports contain arrays of micro-cavities into which crystals are loaded by pipetting a suspension onto the surface and drawing away excess mother liquor via vacuum. The loaded chip is sealed between Mylar films and rastered through the XFEL beam at up to 100 Hz. The opaque MISP variant, made from carbon-black-doped polymer, eliminates light contamination between wells — critical for light-sensitive proteins like CrLOV.
The Nogly Lab team with collaborators at the Cristallina beamline of SwissFEL — two weeks after the European XFEL experiment, with the same CrLOV sample and renewed energy.
Sometimes, if you can’t get the results you hope for, you can simply draw it on a whiteboard — still works! A hand-drawn CrLOV structure with alpha-helices, beta-sheets, FMN chromophore, and Arg58.
After two exciting experiments…
Two XFEL facilities, two different sample delivery methods, and countless hours of round-the-clock teamwork — February 2025 was a month our lab will not soon forget. The data is now being processed and analyzed, and we look forward to sharing the scientific results in due course.
But after the intensity of back-to-back beamtimes, a moment of rest is well deserved.
After two exciting experiments comes time to relax with a view from Uetliberg outside of Zürich. Cheers to science, teamwork, and the mountains!
Are you a structural biologist passionate about time-resolved crystallography, XFELs, and light-driven proteins? We are always looking for talented postdocs and PhD students to join our team. Visit our Join Us page to learn more about open positions at the Dioscuri Center for Structural Dynamics of Receptors.
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