Work Health and Safety
Typical hazards at a microscopy facility
Hazards at a microscopy facility can be broadly categorised into three key areas:
1. Your specimen
2. Microscopy specimen preparation
3. Microscope operation
SITEMAP
- What is Microscopy?
- Why do microscopy?
- Magnification
- Magnification and measurement
- Resolution
- Depth of field
- Electromagnetic Spectrum
- Introduction to the Electromagnetic Spectrum
- Wavelength, frequency and energy
- Electromagnetic spectrum and microscopy
- Interactions with Matter
- Lenses and Aberrations
- Lenses
- Aberrations
- Types of aberration
- Overcoming aberrations
- Calibrations
- Nyquist Sampling
- Detectors and Cameras
- Digital Images
- Introducing digital images
- Images for Print in Journals and Posters
- Images for Presentation
- Colour enhancement
- Image Ethics
- Credits
- What is SEM?
- Background information
- SEM Basics
- Applications and uses of SEM
- What can it do differently to a light microscope?
- What can’t it do?
- How does an SEM work?
- Structure of an SEM
- The electron gun
- How the gun works
- Electron sources
- Filament saturation
- The vacuum system
- Vacuum system overview
- Types of pumps
- Vacuum requirements
- Water chilling system
- Structure of the column
- The specimen chamber
- Beam/specimen interactions
- The image
- How do I get a good image?
- A basic guide to using an SEM
- Specimen preparation
- Accelerating voltage
- Apertures
- Spot size
- Working distance
- Contrast and brightness
- Magnification and calibration
- Scan rate and signal to noise
- Image artefacts and trouble-shooting
- Specialised SEM techniques
- CL
- ESEM
- Cryo-SEM - Cold stage
- FIB
- EDS
- EBSD
- EBL
- Backscatter
- Credits
- What is TEM?
- Introduction to TEM
- Key advantages
- What the TEM can do
- What the TEM can't do
- How does a TEM work?
- Overview of TEM workings
- Instrument design
- Resolution
- Components of a TEM
- Introducing TEM components
- Vacuum system
- Electron gun
- Electron column
- Electromagnetic lenses
- Specimen/sample chamber
- Image capture
- Detectors
- How images are formed
- Image formation basics
- Image types
- Bright-field images
- Dark-field images
- Diffraction
- How do I get a good image?
- Instrument alignment
- Getting started with instrument alignment
- Condenser lens centre
- Condenser lens stigmation
- The eucentric position
- Focus
- Use of objective appertures
- Final adjustment
- Problems with lenses and alignments
- Types of problems
- Spherical aberration
- Chromatic aberration
- Astigmatism
- Understanding instrument settings
- Specimen preparation
- Introduction to specimen preparation
- Specimen holders
- Organic (soft) samples
- Getting started with organic samples
- Ultramicrotomy
- Immunolabelling
- Staining
- Cryo-fixation
- Chemical fixation
- Dehydration
- Infiltration
- Polymerisation
- Grid mounting
- Negative staining
- Cryo-substitution
- Low temperature polymerisation
- Freezing methods
- Cryo-ultramicrotomy
- Cryo-transfer
- Freeze-etch
- Replica
- Inorganic (hard) samples
- Sample prep flow chart
- Powders
- Preparing from the bulk
- Dimpling
- Mechanical polishing
- Electro-polishing
- Ion beam thinning
- Focussed ion beam milling
- Artifacts
- Specialised TEM techniques
- Cryo - TEM
- Introduction to Cryo
- Why Cryo TEM?
- The freezing process
- Diffraction
- Introduction to diffraction
- Image appearance
- The diffracted beam
- Tilting
- Camera length
- Kikuchi patterns
- Selected area diffraction (SAD)
- Ring patterns
- Convergent beam electron diffraction (CBED)
- Dark-field imaging
- High resolution imaging
- What is high resolution imaging?
- Scanning TEM (STEM)
- Bright field STEM
- Beam–sample interactions
- Use of ronchigrams in STEM
- STEM detectors
- High-angle-annular dark-field (HAADF)
- Energy dispersive spectroscopy (EDS)
- Introduction to EDS
- Quantification of EDS data
- Electron energy loss spectroscopy (EELS)
- Credits
- Introduction
- Basics
- What makes an objective good?
- Aberrations
- Light microscopy
- The complete microscope
- Koehler illumination
- The light path and microscope
- Performing Koehler illumination
- Transmitted Light Imaging
- What is Brightfield imaging?
- Microscope components for transmitted light imaging
- Light Path for BF microscopy
- Types of transmitted light imaging
- The five techniques
- Bright field
- Dark field microscopy
- Phase contrast
- Differential interference contrast imaging (DIC or Nomarski imaging)
- Polarised light microscopy
- Reflected light imaging
- Fluorescence microscopy
- What is Fluorescence imaging?
- The light path and microscope parts
- Confocal microscopy
- What is Confocal imaging?
- A practical confocal microscope
- Components of the confocal microscope
- Laser
- Filters
- Photomultiplier tubes (PMTs)
- Practical image acquisition
- Adjustments
- The eternal triangle
- What is important?
- The Confocal Pinhole
- Scanning and resolution
- How scanning works
- Scan areas and relationship to pixels and resolution
- Zoom
- Detection parameters
- Laser power
- Adjusting the Image and Detector Controls
- Averaging
- Sequential and simultaneous imaging
- Using multiple dyes
- Fluorescence Spectra
- Fluorescence Spectral Overlap
- Simultaneous Imaging
- Sequential Imaging
- Collecting Z stacks
- What is a Z-stack?
- Optical Section Thickness
- Nyquist Sampling for Z stacks
- Under and Over Sampling in Z Stacks
- Projections
- Image rotations
- Axial resolution and Optical section thickness
- Super-Resolution Microscopy
- The power of Super-Resolution
- Criteria for optical resolution
- Advantages of higher resolved images
- STED/RESOLFT techniques
- Overview to STED/RESOLFT
- STED
- Introduction to STED
- The Photo-physical Principle
- Resolution in STED microscopy
- RESOLFT
- Single molecule localisation techniques
- One point at a time
- PALM
- dSTORM / GSDIM
- STORM
- PAINT and DNA PAINT
- 3D-SMLM
- Sample preparation for Super-Resolution Microscopy
- General Considerations
- Sample preparation
- Sample fixation
- Properties of labels
- Live-cell Imaging
- Labeling via affinity probes
- Sample preparation for STED microscopy
- Fluorophores and strategies for fixed samples
- Live-cell STED Imaging
- Sample preparation for PALM
- Fluorophores and their properties
- Sample preparation for dSTORM
- Fluorophores and strategies for fixed samples
- Super-Resolution Image Acquisition
- STED
- The optical path of a STED microscope
- STED Imaging
- SMLM
- The optical path of a SMLM microscope
- Imaging strategies for dSTORM
- Blinking fluorophores
- Laser power
- Buffer
- UV Light
- Exposure time
- Image reconstruction from SMLM data
- Credits
- What is Cryo-EM?
- Introduction to cryo techniques
- Why cryo?
- Which cryo technique to use
- Challenges of cryo
- Principles of freezing
- Properties of water
- Freezing of water
- Types of freezing
- Plunge freezing
- High pressure freezing
- Other freezing techniques
- Cryo-TEM
- Introducing cryo-TEM
- The cryo TEM
- The microscope
- Electron energy filters
- Phase plates
- Electron detectors
- Imaging in a cryo-TEM
- How images are formed
- Fourier Transformation
- Why do Fourier transforms
- What is a Fourier Transform?
- Fourier transforms in cryo-TEM image processing
- The power spectrum
- Why do we even need to look at the power spectrum?
- Contrast transfer function
- Single particle analysis
- Introducing Single Particle Analysis
- Biochemical preparation and stabilisation
- Specimen screening by negative staining
- Vitrification
- Optimisation of orientation and distribution
- Specimen screening by cryo
- Data acquisition
- Motion correction
- Dose weighting
- Averaging
- Particle picking
- 2D classification
- 3D reconstruction
- Validation
- Sub-tomogram averaging
- Cryo-tomography (cryo-ET)
- Introducing cryo-electron tomography
- The TEM for cryo-ET
- Phase plates
- Sample preparation
- Data acquisition
- Tomogram reconstruction
- Tomogram interpretation
- Sub-tomogram averaging
- Introducing sub-tomogram averaging
- Particle picking
- Sub-tomogram averaging and alignment
- Sub-tomogram classification
- Model refinement and validation
- Electron crystallography
- Diffraction-based Cryo-EM Techniques
- 2D crystallography
- Introducing Micro-ED
- The sample for Micro-ED
- Sample preparation for Micro-ED
- The TEM for Micro-ED
- Sample screening for Micro-ED
- Data collection – Micro-ED
- Data analysis – Micro-ED
- Cryo-SEM
- Introducing cryo-SEM
- Cryo-SEM design
- Sample preparation – Freezing & cryo-transfer
- Sample preparation – Fracturing and cryo-planing
- Sample preparation – Sublimation
- Sample preparation – Coating
- Cryo-SEM operation
- Cryo-SEM microanalysis
- Cryo-SEM artefacts
- Cryo-FIB
- Introducing cryo-FIB
- TEM lamella production by cryo-FIB
- Cryo-FIB-SEM Volume Imaging
- Cryo-ultramicrotomy
- Credits
- Introduction to XRD
- XRD basics
- Interesting facts
- Nobel prizes for research with X-rays
- Background information
- X-rays Overview
- Properties of X-rays
- Production of X-rays
- The geometry of crystals
- Crystal structure
- Miller Indices
- Principles of diffraction
- Wave structure
- Interaction of X-rays with matter
- Diffraction of X-rays by a crystal
- Penetration depth
- Diffraction measurements
- XRD in practice
- Anatomy of an X-ray diffractometer
- Anatomy of an X-ray diffractometer - Intro
- Source
- Primary optics
- Sample holder & stage
- Secondary optics
- Sample preparation
- Types of samples
- What is good data?
- The importance of specimen height
- Safety
- Analysis of data
- What the data tells you
- Phase identification
- Quantitative powder diffraction
- Quantitative analysis
- Factors affecting peak intensity
- Factors affecting peak intensity - Intro
- Structure Factor
- Multiplicity factor
- Lorentz polarisation factors
- Temperature Factor
- Summation of factors effecting peak intensity
- Factors effecting peak width
- Rietveld Refinement
- What is Rietveld refinement?
- Summary of analysis cues
- Specialist techniques using XRD
- Texture
- What is texture?
- Displaying texture
- Measuring texture with X-rays
- Normalisations
- Residual stress
- Glancing Angle XRD
- Glancing angle XRD
- Credits
- Introduction
- Welcome
- What is microanalysis?
- Background information
- What is energy dispersive spectroscopy?
- Outputs from EDS analysis
- X-ray generation
- Generation of X-rays in the electron microscope
- Bremsstrahlung X-ray generation
- Kramer's law
- Characteristic X-rays
- Characteristic X-ray generation
- Nomenclature
- The X-ray spectrum
- Moseley's law
- X-ray intensity
- Intensity basics
- Fluorescence yield
- X-ray absorption
- X-ray detection
- X-ray detection by EDS
- The detector
- The pulse processor
- The multi-channel analyser or display
- Care and calibration
- EDS spectral resolution
- EDS spectral artefacts
- Qualitative EDS
- Qualitative EDS X-ray microanalysis using SEM and TEM
- X-ray peak identification
- Quantitative EDS
- Quantitative EDS - overview
- Limitations of quantitative analysis
- Standardized quantitative analysis
- Spectral processing
- Concentration calculation
- Accuracy of EDS
- Accuracy, precision and detection limits
- Random and systematic errors
- X-ray mapping
- Mapping information
- Parameters for X-ray mapping
- Artefacts in X-ray mapping
- Credits
- What is APT?
- Background Information
- Overview
- Applications of APT
- What can APT do differently?
- What can't APT do?
- A brief history of APT
- How does APT work?
- Intro to the technique
- The principle of APT
- Two flight path options
- Position of atoms within the sample
- Atomic position in the sample
- Chemical identification
- 3D data visualisation
- Laser-assisted APT
- Spatial resolution of APT
- Mass resolution of APT
- Essential parts of an Atom Probe
- The vacuum system
- Handling and transferring samples
- The local electrode
- Ion detection
- The voltage control system
- The Laser system
- The cryogenic system
- The control system
- How do I get good APT data?
- Specimen preparation
- Specimen requirements
- Two main techniques
- Electropolishing
- Focused Ion Beam (FIB)
- Sample insertion in the atom probe
- Mounting the sample
- Inserting the sample in the atom probe
- Specimen coarse alignment
- Collecting data
- Data quality
- Experimental parameters
- Voltage mode acquisition
- Laser mode acquisition
- End of data acquisition
- Data processing and reconstruction
- Data processing steps
- Selection of ion sequence range
- Selection of region of interest (ROI)
- Time of flight corrections
- Mass calibration
- Ranged-ion assignment
- Reconstruction
- Key reconstruction parameters
- How do I analyse APT data
- Mass spectral analysis
- Concentration space analysis
- Grid voxelisation
- Interfaces
- Proxigram analysis
- Solute analysis / Clustering
- Spatial distribution maps
- Specialised APT techniques
- Field Ion Microscopy
- Correlative SEM / APT
- Cryogenic transfer capabilities
- Credits
- What is FIB?
- FIB overview
- Common applications of FIB
- Ion sources
- Ion–Solid Interactions
- Introduction to ion solid interactions
- How many ions?
- Interaction Overview
- Dislocation/Displacements
- Ion Implantation
- Secondary Electrons
- Secondary Ions, Backscattered Ions and Phonons
- Energy loss
- Collision Cascade
- Sputtering
- Sputtering Overview
- Sputtering Yield
- How does a FIB-SEM work?
- Overview
- Components of FIB
- Introduction to components
- Vacuum system and Chamber
- SEM and FIB columns
- Imaging and Analytical Detectors
- Sample Stage
- Gas Injectors
- Manipulators
- Geometry
- Concepts
- Stage Tilt
- Beam Geometry
- Imaging Artefacts
- Eucentric Height
- Coincidence Point
- Applications
- Ion Beam Imaging
- Image Formation
- Charging
- Channeling Contrast
- Destructive Imaging
- Milling
- Patterning Mechanism
- Patterning Parameters
- Patterning Parameters
- Acceleration Voltage
- Aperture/Beam current
- Passes/Dwell time
- Overlap/Pitch
- Scan Direction
- Beam Alignments
- Artefacts
- Introduction to Artefacts
- Curtaining
- Redeposition
- Implantation
- Phase Transformations
- Amorphization
- Interface Mixing
- Heat damage
- Channeling
- Deposition
- Cross-sectioning
- Introduction to cross-sectioning
- Tips and Tricks
- TEM-lamella preparation
- Intro to TEM-lamella preparation
- Process Steps
- Deposition of protective layer
- Prepare Lamella via cross-sectioning
- J-cut
- Lift-out
- Thinning
- Polishing
- 3D Tomography
- Credits
- Introduction
- AFM
- Background information
- Imaging: Interactions
- What can you measure?
- Imaging modes
- Intro
- Contact mode
- Tapping mode
- Non-contact mode
- Specimen
- Specimen choice
- Contact and tapping modes
- Non-contact mode
- Data display
- Artefacts
- Image artefacts
- Tip artefacts
- Scanner artefacts
- AFM in practice
- Laser alignment
- Laser alignment on cantilever
- Cantilever tuning
- Image quality optimisation
- Scan rate
- Gains
- Set-point
- Contact mode
- Tapping mode
- Feedback
- Control signal
- Control modes
- Discontinuous
- Continuous
- Proportional control (P)
- Integral control
- Derivative control
- Composite control (PID)
- Other Techniques
- Scanning Tunneling Microscopy (STM)
- Near-field scanning optical microscope (NSOM/SNOM)
- Tip enhanced Raman spectroscopy (TERS)
- Credits
- Introduction to SIMS
- What is SIMS?
- Mass analysers in SIMS
- Applications of SIMS
- Basic principles of SIMS
- Ion–Sample Interactions
- Static vs dynamic SIMS
- ToF-SIMS
- Primary-Ion Sources for ToF-SIMS
- Liquid-Metal Ion Guns
- Cluster Sources
- Pulsing
- Bunching
- Collection optics
- Time of Flight analysers – overview
- Inside the analysers
- Inside the ToF analyser
- Reflectron-TOF
- ESA TOF
- Detectors
- Data
- Sample preparation
- Sample requirements for ToF-SIMS
- Powders
- Suspensions and solutions
- Applications of ToF-SIMS
- Typical applications
- Pharmaceutical science
- Geology and mineralogy
- Forensics
- Large Geometry SIMS and NanoSIMS
- Common features
- Common features of the Large Geometry and NanoSIMS
- Primary Ions and Primary column
- Sample chamber and stage
- Dynamic mode
- Secondary column
- Double focussing mass spectrometer - overview
- Inside the mass spectrometer
- Electrostatic analyser
- Magnetic sector analyser
- Collection system
- Magnetic sector analyser operation
- Detectors
- Sample preparation for LG and Nano-SIMS
- LG-SIMS - specific capabilities
- Benefits of LG-SIMS
- Data Output from LG-SIMS
- Instrument performance
- Calibration
- Matrix effect
- LG-SIMS for isotope geochemistry
- Applications of LG-SIMS in geoscience
- LG-SIMS for Nuclear Safeguards and Forensics
- NanoSIMS - high resolution imaging mass-spectrometry
- Benefits of Nano-SIMS
- NanoSIMS applications
- NanoSIMS – Cancer therapeutics
- NanoSIMS – Environmental processes
- NanoSIMS – Medical
- NanoSIMS – Geology
- NanoSIMS – Materials Science
- Credits
- What is research data?
- Introduction to research data management
- Maintaining quality and integrity
- Benefits of managing research data
- FAIR data principles
- What is FAIR data?
- The FAIR principles explained
- Persistent identifiers
- Are FAIR data and open data the same?
- The CARE principles
- What are CARE principles?
- What are Indigenous data?
- The CARE principles explained
- Be FAIR and CARE
- FAIR and CARE are complementary
- Global guidelines
- Research data management plans
- Credits
- Microscopy Concepts
- Scanning Electron Microscopy
- Transmission Electron Microscopy
- Light & Fluorescence Microscopy
- Cryo-Electron Microscopy
- X-ray Diffraction
- Energy Dispersive Spectroscopy
- Atom Probe Tomography
- Focused Ion Beam
- Scanning Probe & Atomic Force Microscopy
- Secondary Ion Mass Spectrometry
- Research Data Management
- Work Health and Safety
