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Welcome to MyScope

Online Modules

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 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

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
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
Typical hazards at a microscopy facility
Your specimen
Microscopy specimen preparation
Microscope operation
Local rules
Supporting documentation
Summary
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

Modules in PDF

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
Work Health and Safety
Research Data Management

Simulators

SEM Simulator
TEM Simulator
LFM Simulator
XRD Simulator
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APT Simulator
FIB Simulator

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