2. Development of nano/micro scale sectioning tools based on charged particle beam for biological systems Jing Fu Sanjay B. Joshi Department of Industrial and Manufacturing Engineering The Pennsylvania State University Jeffrey M. Catchmark Department of Agricultural and Biological Engineering The Pennsylvania State University

3. Background Future biomedical research will rely on “more detailed understanding of the vast networks of molecules that make up our cells and tissues, their interactions, and their regulation” (NIH Roadmap for Medical Researches) Subramaniam, Current Opinion in Microbiology 2005 The development of Electron Microscopy (EM) has bridged a gap between cellular structure and protein structure (Subramaniam, 2005)

4. Background - Bioimaging Electron Microscopy –Scanning Electron Microscopy (SEM) –Transmission Electron Microscopy (TEM) Sample Preparation –Chemical fixation –Resin embedded –Vitrification (Cryo-fixation) Amorphous ice by plunge freezing to <~136K Immobilized instantly for in situ imaging Sectioning by microtome required for bulk samples –Vitrification only applies to sections of µm thickness –TEM requires thickness of several hundreds nanometers

5. Background – Charged Particle Beams Charged Particle Beam (Ion Beam, Electron Beam) –Advancements of Electrostatic optics (ion beam) and Magnetic lenses (e- beam) –Submicron feature patterning Focused Ion Beam (FIB) –Typical based on gallium ions (Ga + ) –Digitally controlled with 3D geometry capability –Larger material removal rate (ion vs. photon, electron)

6. FIB for Biosectioning Frozen hydrated samples (cryo-fixed) –A preliminary study (Heymann, J. Structural Bio. 2006) –Plant cells, E.coli (Marko et al., Nature method, 2007), Mammalian cells (McGeoch, J. Microscopy, 2007) FIB milled Arabidopsis courtesy of Gang Ning at PSU FIB milled E.coli and reconstruction (Nature method, 2007), with permission from Michael Marko at Wadsworth Center

7. Advantages Provide in situ sectioning and imaging of cell structures or systems Fully digitally controlled operations for “Slice and View” by dual beam (FIB/SEM) system and 3D tomography Less distortion or compression vs. conventional microtome Occupational safety (risk of neuropathy using cryomicrotome) Optical Image (scale: 20 μ m)SEM Image (scale: 3 μ m) FIB milled frozen Acetobacter xylinum

8. Ion-Solid Interactions - Overview

9. Challenges Ion-solid interactions –Limited study on ion sputtering in a cryogenic environment –Ultrahigh sputtering rate reported which invalidates conventional models Process control –System Settings: ion energy, ion current, etc. –Process Parameters: temperature, target material, etc. Process characteristics –Surface morphology –Aspect ratio of features

10. Material Removal Rate Y Defined as Sputtering Yield (molecule/ion) or Sputtering Rate (µm 3 /nC) Classical model (For monatomic or alloys materials) –Linear Cascade Collisions (LSS) by Sigmund –Nuclear sputtering dominant Monte Carlo simulation (SRIM/TRIM)

11. Setup FEI Quanta 200 3D DualBeam (FIB/SEM) at Material Characterization Lab, Penn State University Ion Beam –Ga +, 10-30 keV –Beam Spot Size (Minimum 7 nm) Target Samples –Amorphous Solid Water (ASW) by Vapor Deposition –Hyperquenching Glassy Water (HGW) by Plunge Freezing Temperature –83 K – 123K

12. Sputtering Rate of Solid H 2 O Previously limited to astrophysics since early 1980 Ion energy dependent (10 keV – 30 keV Ga + ) –Y=Y n +Y e, Magnitude of 10 µm 3 /nC at 30 keV (~0.3 µm 3 /nC for Si) –Nuclear sputtering Y n  S n –Electronic sputtering Y e  (S e ) 2 J. Fu, S.B. Joshi, J.M. Catchmark, J. Vacuum Sci Tech A, to appear

13. Sputtering Rate of Solid H 2 O Temperature dependent –Y increases with the increase of T –Y(T)=Y(0)(1+αe -β/kT ) Varied energy dissipation

14. Sputtering Rate of Solid H 2 O Incident angle dependent –Maximum of Y at 70 degree –Y(θ)=cos -1.3 Y(0) from 0 degree to 70 degree Energy transfer by Ion closer to the surface Decrease of ion effective volume at high θ

15. Surface Morphology Submicron features developed on ice upon ion sputtering Various morphology, dome/pillar, terraces, etc. Incident angle dependant FIB milled water ice at different incident angle (100 pA, 30 keV, 93 K)

16. Redeposition Sputtered atoms/molecules may reattach to the surface – redeposition May result in significant deviation in geometry Slow milling (400 µs dwell time) on water ice

17. Process Simulation 300 nm trench milled on water ice at 93 K Cylindrical features of diameter 5 µm milled on water ice at 93 K Results of process simulation

18. Closing Remarks Highlights –Development of charged particle beams (ion and possible electron beam) as cryomicrotome for sectioning biological samples –Modeling of ion sputtering (keV Ga + ) water ice –Investigation of process and system variables Future development –Cryo-transfer methods and protocols –Ion interactions with macromolecules and process database –Biological effects for development of cell surgery

19. Acknowledgements Lucille A. Giannuzzi, FEI company Michael Marko, Wadsworth Center Gang Ning, Penn State University Sriram Subramaniam, NIH Use of facilities at the PSU Site of the NSF NNIN under Agreement # 0335765 Cryo-FIB seed fund, MCL, Penn State University

20. Thank you Thin section of ice about 400nm ready for TEM lift out (scale: 5 μm ) Top View FIB milled Ice section 93 K