Cutting-Edge Development in Molecular Biophysics: Advanced Instrumentation Research

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

2. 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

3. 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 …

4. 5 key elements must be under control

5. Sample control and characterization • Understanding the interaction between the laser pulse and the sample To deliver the sample and understand the interaction • 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 Vacuum 10-6 mbar Atmospheric pressure 103 mbar

6. 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.

7. 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

8. 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)

9. Outlook: sample delivery and control 3: Pulse on demand Laser controls Detectors (PMTs) Timing electronics optics Sample injector Imaging laser Tracking lasers diffraction Imaging detector 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

10. 5 key elements must be under control