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Cells & Microscopy
Uni of Notts, Genes Molecules & Cells, first year
| Question | Answer |
|---|---|
| 3 elements of cell theory | 1. All organisms are composed of 1 or more cells 2. Cells are smallest unit of life forming a basis for organisation of more complex organisms 3. Cells can only be created from division or specialisation of other cells |
| Resolution of microscopes + max resolution of light | The minimum distance between which 2 structures can be seen as separate & discreet, for light this is 200nm at 2000x magnification |
| Fluorescence microscopy | Fluorescent dye or protein (typically GFP from jellyfish) is excited by UV light from within the cell & reflects an image using the same mirror as the UV light to give an image of the cell |
| Fluorescence microscopy: super resolution techniques (STED example) | Overcomes the diffraction barrier by using a depletion beam to reduce surrounding fluorescence so only essential structures are illuminated, max resolution of 20-30nm |
| Laser scanning confocal light microscopy | Magnifies thin plane of a thicker sample that doesn't need to be sectioned. Requires fluorescence excited by a laser. Multiple can be taken at different planes to compile a 3D image |
| Phase-contrast microscopy | Light is refracted slightly by living samples & the refractive index difference can be used to highlight intracellular components without toxic stains |
| Differential interference contrast (DIC) microscopy | Changes in refraction index of the cell as a whole can be contrasted at different angles to form a 3D image |
| Atomic force microscopy (AFM) | Visualises surface by using a fine tip point which beams a laser that's reflected back to show distance from the surface which allows the surface of the cell to be visualised in high resolution |
| Sample preparation steps purpose: 1. Fixation 2. Embedding 3. Sectioning 4. Staining | Sample preparation steps purpose: 1. Stops tissues falling apart, usually formaldehyde or acetic acid 2. Providing mechanical support on slide using wax or resin 3. Cutting to 0.5-10μm using metal, glass, or diamond microtome 4. Improving visibility |
| How electron microscopy works | Tungsten electron gun focuses electrons through powerful electromagnetic lenses, they reflect off heavy metal stains (such as osmium or uranium which differentiate samples |
| Cryofixation freeze-fracture | Cells rapidly frozen, splitting down lines of weakness, are coated in heavy metal (platinum normally) to make a replica which can be viewed |
| Freeze-facture steps: 1. Fracturing 2. Etching 3. Shadowing 4. Replicating | Freeze-facture steps: 1. Cutting sample with microtome, usually through phospholipid bilayer 2. Sublimating thin layer of ice on top of sample 3. Depositing thin layers of C or Pt under vacuum over sample 4. Removing shadowed replica for EM viewing |
| Cryo-electron microscopy | Vitrifying solution of cells or biomolecules on a grid to prevent damaging ice-crystals forming before using them in an EM |
| Benefits of cryo-electron microscopy | Avoids leaving artefacts like traditional dehydration & staining would, this allows samples to be viewed in near native state |
Created by:
Beech47
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