Introduction to Light and Fluorescence Microscopy by Sydney Microscopy and Microanalysis

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