Fichas
Microscopy
Fichas del conjunto (13)
- 1Light microscopy principlesMLight focussed onto specimen by lenses in condenser
Objective and eyepiece lenses focus image - 2Limits to light microscopyonly 0.2um resolution due to light wavelength
Cannot see colourless (e.g. much of cell biology) - 3StainingIncrease contrast, however must be killed and preserved as cut sections so not live cells
- 4Phase contest microscopyLive cells can be seen
Phase of wave changes when passes through different density
Contrast created when 2 sets of waves combine - 5Scanning electron microscopyElectron wavelength shorter so higher resolution
Fixing and drying specimen then coating with thin layer of heavy metal e.g. gold
Scattering of electrons creates 3d image
Amount of scattering depends on angle - 6Transmission electron microscopeCells stained with heavy metal salts to create electron dense regions
Electrons pass through unstained regions and focussed to form image
Vacuum prevents electron scattering by air
Cells must be fixed and very thin sections as limited penetrating power
Issues by artefacts - 7Fluorescent and Confocal MicroscopyFluorescent dyes linked to antibodies to label molecules and laser and computer technology activates fluorescence and collect emitted light from single focal plane
optical slice of cell viewed without out-of-focus laters interfering - 8Advantages of theseCan create 3d images
Can following living cells when combined with green fluorescent protein/ tags to locate protein interaction and dynamics
Use FRAP and FRET - 9X-ray crystallographyOrdered crystals scatter x-rays to give diffraction spots - patten given information of atoms via electron density
Can determine protein structure
Optimal conditions required - 10Cell fractionation, centrifugation, sedimentationOpen cells via osmotic shock, ultrasonic vibration or homogenisation, but leave organelles in tact
Spinning separates those differing largely
Sucrose density gradient allows greater separation degree - 11What happens to broken membranes?Release into microsomes
- 12Velocity sedimentationSpeed at which component separates = S value
If sucrose concentrated, sub cellular components stabilise at position where density matched surroundings (equilibrium sedimentation) - 13Mass SpectrometryRoutine technique for protein identification and characterisation
Proteins digested into peptides by protease
Precise mass gives unique fingerprint
Further fragmentation during ionisation gives amino acid sequence
Mass information can identify post-translational modifications (e.g. glycoprotein)
Información
- Descripción
- Biology of Cells IA Fichas sobre Microscopy, creado por Alice Hathaway el 07/01/2019.
- Creado por
- Alice Hathaway
- Visitas
- 12
- Valoración