Research-driven instrument development at the Laboratory of Molecular Biophysics RECONSTRUCTED CELL STRUCTURE Jakob Andreasson Laboratory of Molecular Biophysics Uppsala University ELI-Miniworkshop on Biological Imaging with Intense Ultra-Short X-Ray Pulses Friday 13th April 2012 ∞ 30 60 Resolution length on the detector (nm)

LMB: A decade of experience of “Diffraction before destruction” imaging Femtosecond imaging of delicate structures using ultra-short and ultra-intense X-ray FEL pulses - Circumvent the damage problem by capturing an image before the sample has time to respond Coulomb explosion of a protein molecule by an XFEL pulse 3x1012 photons focused to a 100 nm diameter spot, 12 keV photon energy Lysozyme SHORT PULSE (10 fs) LONG PULSE (300 fs) Ionisation by X-rays modifies atomic scattering factors and the positions of atoms/ions Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. Hajdu, J. (2000) Nature 406, 752-757

Single particle imaging (mimivirus), AMO end-station, CAMP instrument M. Marvin Seibert, et al. Nature 470, 78-81 (03 February 2011) Single mimivirus particles intercepted and imaged with an X-ray laser Recent development …

5 key elements must be under control We would like to test the concept and develop the technologies we need to address several crucial design questions: How can we inject molecules into the beam (in vacuum) so they maintain their natural structures? How short an x-ray pulse is needed to circumvent damage problem? How can we reconstruct 3D structures from many 2D diffraction patterns? How can we record diffracted single-photon counts from a 1012 photon pulse?

To deliver the sample and understand the interaction Sample control and characterization Understanding the interaction between the laser pulse and the sample a.1) Sample delivery For abundant sample aerodynamic lens injection works close to theoretical limits High hit-rate but un-known sample orientation (hits occur at random) Techniques for rare samples would be useful sample on demand (sample trapping) pulse on demand (sample tracking) a.2) Sample characterization Structural integrity and control of sample dynamics during the delivery process Mass spectroscopy techniques (sample selection and characterization) Fluorescence and vibrational spectroscopy, elastic and inelastic light scattering Aerodynamic lens Atmospheric pressure 103 mbar Vacuum 10-6 mbar

Injector pictures Top: Green laser light from an alignment laser scattering of a <50 μm particle beam exiting the injector nozzle. Right: A trace of injected cells deposited on a gel-box substrate Top: Aerosol nozzle ejects a mist of aerosolized sample. A blue laser generates a blue spot where the laser is focused on the droplet stream by the bottom objective. Right: Outside injector assembly. Left to right: Alignment laser Aerosolization chamber Nozzle/skimmer box with viewport, pressure gauge and pumping Relaxation chamber Motorized X-Y-Z translation stage.

Outlook: sample delivery and control 1 Mass spectrometry-based sample delivery and analysis Pros: - Fairly similar to commercially available high mass spectrometers - Can be coupled to “pulse on demand” Possible cons: Randomness and sample must be charged

Outlook: sample delivery and control 2 Sample on demand: Droplet on demand coupled with in-vacuum optical trapping (low rep. rate) Optical trapping in air and vacuum www.microdrop.de Merging of droplets NATURE PHYSICS | VOL 7 | JULY 2011 | Tongcang Li, Simon Kheifets and Mark G. Raizen* Center for Nonlinear Dynamics and Department of Physics, The University of Texas at Austin, Austin, Texas 78712, USA. M. Horstmann et al. Lab on a Chip, 12, 295 (2012)

Outlook: sample delivery and control 3: Pulse on demand Tracking lasers Detectors (PMTs) Timing electronics Imaging laser Imaging detector optics Laser controls diffraction Sample injector Sample tracking coupled with pulse on demand Performed by M Franck et al. at Livermore in Bio Aerosol Mass Spectroscopy (BAMS) experiments Can even track on fluorescent signal to trigger only on droplets containing a certain sample Anal. Chem. 2003, 75, 5480-5487

5 key elements must be under control We would like to test the concept and develop the technologies we need to address several crucial design questions: How can we inject molecules into the beam (in vacuum) so they maintain their natural structures? How short an x-ray pulse is needed to circumvent damage problem? How can we reconstruct 3D structures from many 2D diffraction patterns? How can we record diffracted single-photon counts from a 1012 photon pulse?