MyScope Train for advanced research Module Contents Module Contents Menu Module Contents Menu MODULE PDF TAILOR THIS MODULE SHOW ALL MENU ITEMS ACKNOWLEDGMENTS Microscopy Australia Facilities Partners Educational Supporters SIMULATOR Publications on MyScope Attribution-Share Alike Unless otherwise noted, content on this site is licensed under the Creative Commons Attribution-ShareAlike 4.0 International License. We cannot revoke these freedoms as long as you follow the license terms. SITEMAP Online Modules Microscopy Concepts 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 Scanning Electron Microscopy 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 Transmission Electron Microscopy 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 Beamsample 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 Light & Fluorescence Microscopy 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 Cryo-Electron Microscopy 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 X-ray Diffraction 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 Energy Dispersive Spectroscopy 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 Atom Probe Tomography 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 (R…