Poster Presentation 24th Australian Conference on Microscopy and Microanalysis 2016

Dual beam microscopy of biological samples (#293)

Annalena Wolff 1 , Yinghong Zhou 2 , Jinying Lin 2 3 , Shifeier Lu 2 , Zhibin Du 2 , Chaturanga D Bandara 2 4 , Yong Y Peng 5 , John AM Ramshaw 5 , Yin Xiao 2 4
  1. Central Analytical Research Facility, Queensland University of Technology (QUT), Brisbane, QLD, Australia
  2. Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), Brisbane, QLD, Australia
  3. Department of Implantology, Xiamen Stomatological Research Institute, Xiamen Stomatological Hospital, Fujian, China
  4. Science and Engineering Faculty, School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology (QUT), Brisbane, QLD, Australia
  5. Manufacturing, CSIRO , Bayview Avenue Clayton, Victoria, Australia

Dual Beam technology, in which a Scanning Electron Microscope (SEM) and a Focused Ion Beam (FIB) are combined in a single device, has established itself as common tool for microanalysis, nanostructuring and in-situ TEM lamella preparation (1). Today, Dual Beams are most commonly used in the material science sector and semiconducting industry (1). Throughout the past decade, this technique has found increasing interest in the life sciences (1-7) due to the Dual Beams unique capability of revealing internal sample structures at high resolution and to prepare TEM-lamellas (sections) at precisely selected points within the sample which cannot be achieved by other techniques. However, artefacts and appropriate parameters for biological samples which limit process induced artefacts and sample damage are not yet well understood. 

This study focusses on Dual Beam Microscopy of (fixed and stained) biological samples. Lift-outs and cross-sections of a variety of embedded and non-embedded biological samples including porcine collagen, a dragonfly wing and dental implants were prepared. With the choosing of appropriate Dual Beam parameters, it was found that embedding is not required. The issues of beam damage such as melting, surface damage and how to avoid it are discussed. Case studies illustrate the difficulties which arise during the TEM-lamella preparation due to the samples 3D structure, and how these can be addressed.  The study shows that non-embedded biological samples can successfully be prepared using Dual Beams if appropriate parameters are selected.

The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Central Analytical Research Facility, Queensland University of Technology as well as the facilities (FEI Scios), and the scientific and technical assistance of Dr Hui Diao, of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.

  1. Drobne et al., ‘Surface Damage Induced by FIB Milling and Imaging of Biological Samples is Controllable’; Microscopy Research and Technique 70; 895-903 (2007)
  2. Leser et al., ‘Focused ion beam (FOB) / scanning electron microscopy (SEM) in tissue structural research’; Protoplasma 246 (1-4); 41-48 (2010)
  3. Earl et al., ‘Characterization of dentine structure in three dimensions using FIB-SEM’; Journal of Microscopy 240, Pt 1, 1-5 (2010)
  4. Milani et al.; ‘How to study biological samples by FIB/SEM?’; Modern Research and Educational Topics in Microscopy, FORMATEX; 148-154 (2007)
  5. Villinger et al.; ‘FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells’; Histochem Cell Biol, 138; 549-556 (2012)
  6. Schneider et al.; ‘Serial FIB/SEM imaging for quantitative 3D assessment of the osteocyte lacuna-canalicular network’; Bone 49, 304-311 (2011)
  7. Stokes et al.; ‘ A New Approach to Study Biological and Soft Materials Using Focused Ion Beam Scanning Electron Microscopy (FIB/SEM)’; Journal of Physics: Conference Series 26; 50-53 (2006)