1. 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)

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,

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

4. 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?

5. 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

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
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
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,

10. 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?