Poster Presentation 24th Australian Conference on Microscopy and Microanalysis 2016

Interleaving patterning method to reduce focused ion-beam induced damage in beam sensitive materials (#218)

Jisheng Ma 1 , Amelia Liu 1 2 , Charlene Janion-Scheepers 3 , Viola Oorschot 4 , Peter Miller 1
  1. Monash Centre for Electron Microscopy (MCEM), Monash University, Clayton, VIC, Australia
  2. School of Physics and Astronomy, Monash University, Clayton, VIC, Australia
  3. School of Biological Sciences, Monash University, Clayton, VIC, Australia
  4. The Clive and Vera Ramaciotti Centre for Structural Cryo-Electron Microscopy, Monash University, Clayton, VIC, Australia

Most ion-beam sensitive materials, such as biological samples and polymers, experience significant heat damage during Focused Ion Beam (FIB) milling processes1 apart from other undesirable effects such as ion implantation and amorphization. The heat generated by the energy loss of the ion beam can cause dramatic local temperature increases in a material with low thermal conductivity2. Cryogenic conditions do not necessarily prevent or delay the degradation of organic samples caused by the local heating3.

An Interleaving Patterning (IP, called interlacing patterning in literature) strategy4,5 was first used for electron beam deposition to eliminate proximity effects caused by consecutive deposition4. Later it was used to eliminate thermal stress during FIB processing5 . Recent development work was focused on improving pattern acuity in nanofabrication6.

The IP technique is extremely effective in preventing local temperature spikes during FIB processing. In IP subsequent dwell points do not overlap, and thus temperature increases are not additive.  Heat dissipates before the ion beam return to modify an area twice.  A trial on a polymer film showed that rapid milling and large area cross-section can be achieved without visible heat damage. Here we employ the IP technique on biological materials.  IP FIB milling was used to cross-section freeze-dried Collembola (springtails) to study the effect of climate change on their cuticular morphology. The specimen morphology was retained during FIB milling and the cuticule ultrastructure7 was revealed. Further experiments on cross sectioning and TEM lamella lift-out of a fixed mouse hypothalamus specimen confirmed that specimen morphology is well retained and that ultrastructure can be imaged.

The IP technique is able to retain specimen ultrastructure, increase contrast and improve resolution, even for biological specimens without chemical fixation and staining. Further opportunities for high-quality specimen preparation will be possible when IP is integrated into the control software in a Cryo-FIB.

The author acknowledges the use of the facilities at the Monash Centre for Electron Microscopy and the assistance of Assoc. Prof.  Matthew Wayland.

  1. Schmied R, et al, Phys. Chem. Chem. Phys., 2014, 16, 6153-6158.
  2. Orso S, et al, Degree Dissertation of University of Stuttgart, 48, Dec. 2005.
  3. Sezen M, et al, Phys. Chem. Chem. Phys., 2009, 11, 5130–5133.
  4. Planck H, et al, ACS Nano, 2012, 6(1), 286-294.
  5. Schmied R, et al, RSC Adv., 2012, 2, 6932-6938.
  6. Orthacker A, et al, Phys. Chem. Chem. Phys., 2014, 16:1658-1666.
  7. Nickerl J, et al, J. R. Soc. Interface, 11:20140619, http://dx.doi.org/10.1098/rsif.2014.0619.