freeze fracture/ etc Flashcards
Why is the resolution of a light microscope limited?
The resolution is limited by the wavelength of visible light. Increasing magnification results in a blurred image, making the maximum magnification typically X400.
How do electron microscopes achieve higher resolution than light microscopes?
Electron microscopes use beams of electrons, which have a much shorter wavelength than visible light, allowing for much higher resolution, up to 1,000,000x magnification.
What do light microscopes reveal compared to electron microscopes?
Light microscopes reveal cell structure, while electron microscopes reveal cell ultrastructure, showing details at a much higher resolution.
What are some disadvantages of electron microscopes?
- Black & white images (color is added artificially).
- The preparation methods kill cells.
- The vacuum inside and electron beams are destructive, killing the cells.
What is an advantage of light microscopes over electron microscopes?
Light microscopes can be used to examine living cells and produce color images.
What are fluorescent stains used for in microscopy?
Fluorescent stains help visualize cellular components that are colorless. These stains absorb light and re-emit it at a longer wavelength, producing colored images (e.g., blue light from UV absorption).
What is immunofluorescence?
Immunofluorescence is a technique where antibodies bound to specific antigens are tagged with fluorescent markers. This allows for multicolored fluorescent images that show the location of specific proteins or other molecules in cells.
What is freeze-fracture electron microscopy used to study?
Freeze-fracture electron microscopy is used to produce detailed images of surfaces within cells, particularly the membranes.
Describe the process of freeze-fracture electron microscopy.
- Rapidly frozen in liquefied propane.
- Fractured with a steel blade at the cell’s weakest points (like membranes).
- Etched by vaporizing some ice.
- Coated with platinum or carbon to create a replica.
- The replica is examined under an electron microscope to reveal cell membrane structures.
Why is freeze-fracture electron microscopy particularly useful for studying membranes?
This technique highlights the phospholipid bilayer of membranes, revealing their internal structure, which led to a significant shift in how membrane structure is understood.
What is cryo-EM (cryogenic electron microscopy)?
Cryo-EM is used for studying the structure of proteins. A thin layer of protein solution is flash-frozen to preserve its natural state and prevent water crystal formation.
How does cryo-EM work?
- A protein solution is applied to a grid.
- The grid is flash-frozen to create smooth, vitreous ice.
- The grid is placed in an electron microscope, and electron detectors record the transmitted patterns of the protein molecules.
- Multiple images from random orientations are combined to create a 3D structure using computational algorithms.
Why is cryo-EM revolutionary for studying proteins?
Cryo-EM can study proteins in various functional states as they freeze, allowing researchers to study proteins that undergo conformational changes during their function, like pyocin.
What is the current resolution capability of cryo-EM?
Cryo-EM can achieve resolutions of up to 0.12nm, allowing the imaging of individual atoms within proteins or other molecules.
How many protein structures are available in the EMDB (Electron Microscopy Data Bank)?
Over 10,000 protein structures are shared in the EMDB as a result of advances in cryo-EM.
