A six part lecture series by Sydney Microscopy and Microanalysis, a Microscopy Australia facility, on the fundamentals of light and fluorescence microscopy. The lectures cover:

Intro to Light Microscopy 1: Microscopy Basics

  • Basic light microscope components
  • Brightfield imaging and related modalities
  • Basic sample preparation for brightfield microscopy
  • Key terms and considerations

Intro to Light Microscopy 2: Principles of Fluorescence Microscopy

In this module you will learn about fluorescence and how it is used to generate high contrast microscopy imaging.

  • Properties of light
  • Absorption and emission and how these relate to contrast and fluorescence techniques
  • The mechanism of fluorescence
  • A Jablonski diagram and how it shows the mechanism of absorption and emission of light by molecules
  • Stokes shift
  • Mechanism of quenching
  • How filters work

Intro to Light Microscopy 3: Actual Microscopes

  • Part 1 – Widefield Fluorescence Microscopes
    • Light Path of a Widefield Fluorescence Microscope
    • Light Sources of a Widefield Fluorescence Microscope: Advantages and Disadvantages
    • Filters, Filter Cubes, Filter Wheels
    • Objective lenses: relevant information written on the barrel (and not written on the barrel!)
    • Magnification versus Resolution
    • Digital Cameras: CCD versus CMOS and Colour versus Monochrome
    • Widefield versus Confocal Fluorescence Microscopy
  • Part 2 – Confocal Microscopes
    • Light Path of a Confocal Fluorescence Microscope
    • Lasers and controlling the laser power
    • Filters/AOBS
    • Scanning and Digital Zoom
    • Nyquist Sampling, Pixel Size, and Pixel Number Point Spread Function and Lateral versus Axial Resolution
    • 3D Image Stack Point Spread Function and Lateral versus Axial Resolution
    • Point Spread Function and Lateral versus Axial Resolution
    • Refractive Index Matching
    • Objective Correction Collars
    • Pinhole and Airy Unit
    • Detectors versus Cameras
    • Spectral Overlap and Simultaneous versus Sequential Scanning
  • Part 3 – Advanced Microscopy Techniques
    • Multiphoton Microscopy
    • Spinning Disk Confocal
    • Super Resolution
    • Total Internal Reflection Fluorescence

Intro to Light Microscopy 4: Sample Preparation and Artefacts

  • Sample Preparation Definition
  • General Workflow: Sample Collection
  • Fixation and Fixative
  • Processing, Embedding and Sectioning
  • Staining
  • Mounting and Storage
  • Artefacts and how to identity
  • Cause of artefacts
  • How to avoid artefacts

Intro to Light Microscopy 5: Fluorescence Microscopy Specimen and Imaging Tricks

  • What is fluorescence?
  • Why do we use fluorescence microscopy?
  • Fixed cell imaging
  • 8 tips and tricks to improve your IF protocol
  • Fixation methods
  • Aldehyde-based fixatives
  • Organic solvents-based fixatives
  • Permeabilisation
  • Blocking
  • Primary and secondary antibody incubation
  • Antibody dilution and cell density optimisation
  • Controls and counterstains
  • Mounting
  • Fixed cell imaging vs live-cell imaging
  • Requirements for non-invasive live-cell imaging
  • Benefits of using low levels of expression of a exogenous fluorescent reporter

Intro to Light Microscopy 6: Digital Image & Data Analysis

  • What is Image Analysis
  • Image Processing Steps
  • Image analysis Packages
  • A Brief History of Digital Images
  • Sampling
  • Quantization
  • Bit Depth
  • Colour Space – CMYK vs RGB
  • Compression in Images
  • Image File Formats
  • Analytical and Visualisation Software in More Detail
  • Collection & Analysis Considerations
  • Real World Examples of Image Analysis