10 Questions
What is a limitation of TEM and SEM that prevents the observation of living specimens?
The requirement of a vacuum environment
What is the primary purpose of cell fractionation?
To isolate and study specific organelles
What is the purpose of using a cold, buffered solution with the same water potential as the cells in cell fractionation?
All of the above
What is the order of sedimentation in differential centrifugation?
Nuclei, mitochondria, then other organelles
What is the term for the fluid that remains at the top of the tube after centrifugation?
Supernatant
What is the primary reason why light microscopes have a limited resolution of 0.2um?
The wavelength of light
What is the purpose of the vacuum environment in an electron microscope?
To prevent particles in the air from deflecting the electrons
What determines the darkness of an area on an electron micrograph produced by a transmission electron microscope?
The areas that absorb the electrons
How does a scanning electron microscope produce a 3D image of a specimen?
By scattering a beam of electrons across the surface of the specimen
What is the formula to calculate the magnification of an image as seen through a microscope?
Magnification = size of image / size of real object
Study Notes
Microscopes
- There are two main types of microscopes: Light Microscopes and Electron Microscopes
- Light Microscopes use convex glass lenses to resolve images 0.2um apart, limited by the wavelength of light
- Electron Microscopes can distinguish between items 0.1nm apart, much higher resolution than Light Microscopes
- Magnification of an image can be calculated using the equation: Magnification = size of image / size of real object
- Resolution is defined as the minimum distance apart that two objects can be distinguished as separate objects in an image
Electron Microscopes
- Two main types: Transmission Electron Microscopes (TEM) and Scanning Electron Microscopes (SEM)
- Electron Microscopes work similarly to Light Microscopes, but use a beam of electrons focused by electromagnets in a vacuum environment
- Vacuum environment is needed to prevent particles in the air from deflecting the electrons
- TEM: a beam of electrons passes through a thin section of a specimen, areas that absorb electrons appear darker on the electron micrograph
- SEM: a beam of electrons passes across the surface and scatters, building up a 3D image depending on the contours of the specimen
- Limitations of Electron Microscopes:
- Whole system must be in a vacuum, so living specimens cannot be observed
- Complex staining process required, which may introduce artefacts into the image
- Specimens have to be very thin, particularly for TEM, for electrons to pass through
- SEM has a lower resolving power than TEM, but both have greater resolving power than a light microscope
Cell Fractionation and Ultracentrifugation
- Cell Fractionation: process of separating different parts and organelles of a cell to study in detail
- Most common method: differential centrifugation
- Steps of homogenization:
- Blend cells in a homogenizer, forming a homogenate
- Spin at slow speed, and heaviest organelles (nuclei) sediment to the bottom
- Remove supernatant and spin at faster speed, next heaviest organelles (mitochondria) sediment
- Repeat process, increasing speed each time to separate next heaviest organelle
Cell Structure
- All living organisms are made of cells, with different types sharing common features
- Humans are made up of different types of cells
Learn about the different types of microscopes used to study cells, including light microscopes and their limitations, as well as electron microscopes and their higher resolution capabilities.
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