CMM Techniques
Advanced Sample Preparation for Transmission Electron Microscopy
Previous EM experience is required.
Theory of freezing hydrated samples for electron microscopy, high pressure freezing, plunge freezing of particle suspensions, handling of frozen hydrated samples, freeze substitution.
Variable: at least 12 hrs plus individual practice.
It should be recognised that these techniques are time-consuming.
Cryo Scanning Electron Microscopy
Previous SEM experience/ training required.
Operation of dedicated cryoSEM. Topics covered routinely include: CryoSEM setup and shutdown; sample preparation and mounting techniques; sample freezing (nitrogen slush plunge freezing); sample fracture techniques; specimen sublimation; sputter coating; sample storage and retrieval; energy dispersive spectroscopy (EDS); and artefacts.
Additional training is available for a high pressure freezer and a freeze drier.
3 hrs theory and 12 to 15hrs of practical.
Cryo Transmission Electron Microscopy
Previous TEM experience is required.
Handling of frozen hydrated samples, use of cryo-sample holders, insertion of cryo-samples into the microscope, low dose electron microscopy.
Variable: at least 12 hrs plus individual practice.
It should be recognised that these techniques are time-consuming.
Electron Backscatter Diffraction (EBSD)
Previous SEM experience/ training required.
SEM training on the JEOL 6610 is a pre-requisite. Basic operation of EBSD. Topics include: overview, sample preparation, EBSD system, collection of crystallographic orientation data and analysis using Oxford Azetc software.
12 hrs.
High Resolution Scanning Electron Microscopy
Previous EM experience is required.
Operation of SEM at a high level. Topic includes: advanced sample preparation, sample loading, electron lenses, microscope alignment, including alignment of the gun, condenser lenses, beam tilt, secondary and back scattered electron imaging, focussing the image, correction of objective astigmatism and digital image acquisition.
10 hrs including practice.
High Resolution S/TEM - HF5000
*Requires >20 hours independent experience on HT7700 or equivalent TEM experience.
For clients requiring aberration corrected STEM, atomic resolution analytical tools (EDX, EELS or EFTEM), or other TEM imaging not available on the HT7700 (including various in situ TEM techniques), training is available on the HF5000. Details of the HF5000’s capabilities can be found here. Training is provided one on one for each client and is tailored for the techniques required for their research.
Training length: depending on training needs this can be from 8-20 hours over a few weeks.
Light Microscopy
No previous experience is necessary.
Practical operation of available light microscopes at a basic level including compound, stereo and confocal microscopes. Topics include: Brightfield field, Differential Interference Contrast (DIC), fluorescence, effects of light, resolution, digital image acquisition, system calibration, sample loading, and sample preparation.
Training length: 3 hours.
Microprobe
Basic SEM training required.
Technical operation of WDS Microprobe for quantitative analysis, mapping, and image acquisition. Advice on sample preparation: resin embedding, polishing and coating. Previous SEM experience/ training required, Plus completion of microanalysis theory.
18 hrs plus individual practice.
Microanalysis Theory - Microanalysis for SEM and Microprobe (EDS/WDS) and TEM
Previous SEM/TEM experience/training required.
Provides an introduction to analysing the elemental composition of samples. Topics include: X-ray production, energy dispersive spectroscopy (EDS), wavelength dispersive spectroscopy (WDS), spectral artefacts and an introduction to matrix correction effects (ZAF).
8 hrs of theory and tutorial (across 3 days).
Negative Staining
No previous experience is necessary.
Practical introduction on how to prepare suspensions for the analysis of small and thin particles (usually thinner than 1µm) for Transmission Electron Microscopy. This is achieved by adsorbing the particles in suspension onto a formvar coated TEM grid, then embedding this sample in a thin film of amorphous heavy metal salts. Training includes principles and how to troubleshoot.
Training length: 2 hours including practice.
Required equipment: forceps, formvar and carbon coated TEM grids.
Sample Preparation for Biological Sciences, Polymer Sciences and General Morphology
Room temperature
No prior experience necessary.
Sample preparation includes: fixation, post-fixation, contrasting and resin embedding, both on bench and using the processing microwave oven.
Training length: 3 hours plus follow up supervised session as necessary.
Cryo/low temperature
No prior experience necessary, however some level of experience in EM is useful.
Tokuyasu preparations including gelatin embedding of biological specimens for low temperature sectioning.
High Pressure Freezing (HPF) and Freeze Substitution (FS): including theory of freezing hydrated samples for electron microscopy, practical use of the high-pressure freezer and subsequent freeze substitution to bring the sample from liquid nitrogen temperatures to room temperature.
Training length: 3-6 hours plus follow up supervised sessions as necessary.
NOTE: it should be recognised that these techniques are time-consuming.
Scanning Electron Microscopy (SEM) for Biological and Physical Sciences
No previous electron microscope (EM) experience is assumed.
Operation of SEM up to an intermediate skills level. Topics include: sample loading, secondary and back scattered electron imaging, effect accelerating voltage has, depth of field, resolution, focusing, astigmatism and digital image acquisition. Sample preparation includes: sample mounting, sputter coating with metals, carbon coating, freeze drying, fixation, chemical dehydration, critical point drying.
This basic training is a prerequisite for upgrade to other SEMs and related techniques e.g. microprobe, ultrahigh resolution FEG, LaB6 cryoSEM, variable pressure (VP) SEM, energy dispersive spectroscopy (EDS), electron backscatter diffraction (EBSD), use of Alicona MEX 3D software.
23 hrs over a two week period.
Subsequent upgrades for other SEMs and related techniques are 5 - 20 hours.
Sample Preparation (physical science) for SEM and TEM
No previous experience is needed.
Cutting with an abrasive disc, hot/cold mounting, manual or automatic polishing, dimple grinding, ion milling, depositing on various TEM grids.
Required equipment: abrasive disc, forceps, TEM grids, grid box.
Training length: depending on the required sample preparation method from 1 hour to 2 hours.
Related Instruments: link Struers CitoVac, Struers CitoPress-15, Struers Accutom-50, Tegramin-20, Gatan PIPS
Sample preparation for SEM
- Cutting on Struers Accutom 50 with SiC/Al2O3 or diamond disks depending on material hardness.
- Hot mounting in conductive or nonconductive bakelite or thermoplastic resin on Struers CitoPress-15 (25 mm or 30 mm diameter cylinders).
- Cold mounting in epoxy resin in CitoVac unit (various mould shapes).
- Semi-automatic grinding and polishing on Struers Tegramin-20 machine with a set of SiC papers of different roughness, followed by fine polishing on cloth disks with diamond suspensions, OPS is also available.
- Chemical etching is not conducted at CMM, but small sample areas can be subjected to ion milling to reveal the surface topography.
Training length: 2 hours including practice.
Sample preparation for TEM
- Manual polishing of metal or ceramic foils on SiC papers till approx. 250 micron thickness, cutting of 3 mm disks, manual polishing to approx. 120 micron thickness, dimple grinding followed by ion milling on Gatan PIPS, OR cutting of approx. 20x20 micron lamellas on SEM-FIB; or
- Deposition of powder samples and nano-particles on TEM grids with various mesh size and support film type (continuous carbon, holey carbon, lacey carbon/formvar); or
- Embedding small (approx. 1x1 mm) section in resin block and cutting thin section with diamond knife using ultramicrotome.
Training length: 1- 2 hours not including practice.
Transmission Electron Microscopy (TEM) – Basic Training
No prior TEM experience necessary.
Operation of TEM at a basic level. Topics covered include: sample insertion, generation of beam, condenser system, electron lenses, aperture insertion, microscope alignments, image formation, correction of astigmatism, the vacuum system and contamination, imaging parameters and digital image capture. Introductory TEM training course.
Required equipment: your samples prepared on TEM grids**
**If you need assistance with this, contact a member of staff.
Training length: 6 hours self-directed learning and theory lecture PLUS 6 hours practical microscope training. This is covered over a 2 week period.
Transmission Electron Microscopy (TEM) – Upgrade Training
EDS/Diffraction/STEM
Minimum 10 hours independent microscope operation is required.
In addition to the basic training, further training includes: theoretical introduction to the beam-sample interaction, generation of X-rays as a result of this interaction, electron diffraction, advanced imaging concepts (bright and dark field imaging), generation of Kikuchi lines and diffraction patterns, scanning transmission mode of imaging, EDS operation software, chemical analysis using EDS (point and map acquisition modes, quantitative determination of chemical composition).
Requirements: minimum 10 hours of independent TEM operation in image mode, SEM-EDS experience is very desirable. Samples prepared on TEM grids by drop casting or by SEM-FIB or thin electron transparent foils prepared by mechanical polishing and PIPS (if you need assistance with this, contact a member of staff).
Training length: depending on the previous experience and training needs, electron diffraction training takes 2-4 hours of individual practical training, including one hour of theory course; STEM/EDS training takes 3-6 hours of individual practical training, including one hour theory course.
Transmission Electron Microscopy (TEM) for Biological Sciences, Polymer Sciences and General Morphology
No previous EM experience is assumed.
To perform TEM on these sample types they need to be prepared in a certain way. For biological samples this means getting from a fixed hydrated state to resin embedded; for polymers they can be directly sectioned or embedded in resin (see Sample Preparation Training). Samples are then sectioned at the ultramicrotome (see Ultramicrotomy training) to produce thin sections that are collected on a TEM grid for imaging (see Basic TEM training).
Step 1: Sample Preparation or Negative Stain
Step 2: Ultramicrotomy
Step 3: Basic TEM training
Transmission Electron Microscopy (TEM) for Physical Sciences
No previous EM experience is assumed.
To perform TEM for physical sciences, the samples need to be prepared according to these minimal requirements: fit into the TEM sample holder (3mm diameter); have electron transparent thickness over a large area; be preserved in the original state of the material (be free of artefacts); be clean and free of contamination; contain detail of interest visible. For powders <100 nm, sample preparation will include mixing powder with a solvent (e.g. ethanol or water), sonication and then depositing a drop of a sample onto a TEM grid. For bulk samples and thin films (metals/alloys, semiconductors, ceramic/insulators) preparation usually consists of multiple steps which may include cutting, resin embedding, ultramicrotomy or mechanical polishing, ion milling or Focus Ion Beam milling (FIB) (see Sample Preparation).
Step 1: Sample Preparation
Step 2: Ultramicrotomy
Step 3: Basic TEM training
Step 4: Upgrade TEM training
Tomography
Previous TEM experience is required.
Operating electron microscopes at an advanced level, imaging with high-end CCD-cameras, acquiring tilt-series of bilological/material samples using Serial EM, computing of tomograms using IMOD, basic segmentation of tomographic data.
Variable depending on research project.
It should be recognised that this technique is extremely time-consuming.
Ultramicrotomy
Room temperature ultramicrotomy
No previous experience is necessary.
Operation of the ultramicrotome for resin embedded or polymer samples to collect thick (500nm) to thin (60-90nm) sections. Also includes associated skills: knife making, aligning and trimming of blocks, picking up sections, staining and troubleshooting.
Required equipment: forceps, TEM grids, grid boxes.
Recommended equipment: Diamond knife.
Training length: 6 hours including practice.
Related Instruments: link
Cryo-ultramicrotomy
Previous sectioning experience/training helpful but not necessary.
Operation of cryo-ultramicrotome for biological (Tokuyasu gelatin embedding) or polymer samples (resin embedding) to collect 60-200nm sections. Also includes associated skills: knife making, trimming of blocks, picking up sections, staining and troubleshooting.
Required equipment: forceps, TEM grids, grid boxes.
Recommended equipment: Diamond knife.
Training length: 6-9 hours including practice per individual (extended if no previous experience on ultramicrotomes for resin sectioning).
Related instrruments: Leica Ultracut UC6 and UC7
Variable Pressure Scanning Electron Microscopy
Previous SEM experience/ training required.
Operation of variable pressure (VP) SEM. Topics include: theory and practical use of SEM chamber pressure to control specimens charge up; the imaging (backscattered and secondary) and X-ray analysis (EDS) of non-conductive samples in the VP SEM; freeze drying specimens using the VP SEM; artefacts and limitations of VP SEM.
5 to 6 hrs practical including some theory.
X-ray Diffraction (XRD)
No previous experience required but Queensland Health Use Licence is a pre-requisite (we can advise on obtaining license but be aware it takes about 3 months).
Operation of X-Ray Diffractometer in powder diffraction mode. Topics include: sample preparation, sample loading, creation of parameter file, creation of job file, processing of traces using Evaluation software, search match using PDF-2 Database, file conversion.
12 hrs.