Biomolecule Visualisation

• X-rays pass through low density materials - bounce off high density materials

• Tune - more gentle x-rays for breakable/defined things e.g. flowers

• Monochrome images
  
• Not all tissues show
• Damage tissues

• X-ray images from multiple angles
• 3D computer model taken

• Virtual dissections
• Zoom in and out
• Background removed - fossils

• Proton-based
• Picks up moleules with magnetic nuclei = NMR fingerprint
• Peaks = different chemical groups

• Type of NMR (less scary name)
• Fluid-filled parts of body picked up

• Live specimens in real time - follow body occurances
• Scan slices - create animation/3D model
• Combine MRI (soft tissues) and CT scans (hard tissues) = more info

• Shine light through specimen
• Brightfield (cells colourless) or darkfield
• Phase contrast - cell edges
  • Histology stain - ratio of colour depends on tissue
    • Haemotxylin staining (blue) = nuclei
    • Eosin staining (red/pink) = everything else
  • Gram stains - detect bacteria
    • Gram +ve - crystal violet
    • Gram -ve - pink counterstain
  • Tissue specific dyes - alizarian red (bone)/alician blue (cartilage) = visualise embryonic development
  • Chromosome specific dyes - map of chromosome arrangement

• Laser-based light microscopy/fluoresence

• Thin optical sections - sharp images
• No backgrond - only sample glowing

• Higher magnification
• Grayscale images
• Dead specimens

Scanning Electron Microscopes (SEM)

• Surface with electrons - bounce back

Transmission Electron Microscopes (TEM)

• Electron beam through ultra-thin specimen

• Absorb at one wavelength and emit at a longer one
• Differenet fluorphores emit different colours = multiple colours
• Subcellular stains - specialised strains
  • DAPI - nuclei
  • Mitotracker red - mitochondria
• Uses
  • Live/dead strains
  • Healthy/infected cells
  • Visualise cytoskeleton
  • Ca2+ actiated fluorphore = calcium waves in glia/fertilisation
  • Indirect probes - cancer shows red under UV = tumor development
  • Embryo development
  • Brainbows

• Can't take photos
• EM = fuzzy images
• Hard to determine shape of 3D molecules
• Hard to display 3D in 2D

X-Ray Crystallography

• Protein crystallised - bombarded with x-rays
• Deflects x-rays different direction
• 2D diffraction pattern -> 3D map
• Shape and order known -> electron density map can be made
  • Hydrogen not detected = too small and too weak charge

NMR

• Solution of pure protein through NMR machine = 2D NMR spectrum
• Converted to 3D structure at atomic level
• Flexible protein - solution and not crystal = range of possibilities for side chain arrangement

Molecular surface - real shape of molecule shown

Spacefill - every atom shown as sphere

Secondary structure (cartoon) - a-helicases and b-sheets shown

Backbone - shows C and N - no side chains

Always pick best representation style for use