3.2.1.3 METHODS OF STUDYING CELLS + MICROSCOPES Flashcards
Principles of optical microscopes:
- can view live specimens
- easy to use
- cost- effective
- can view specimens real colour
Issues with optical microscopes:
- low resolution
- limited magnification
- not all cellular organelles can be observed
Principles of transmission electron microscopes:
- high resolution
- high magnification
- imaged formed upon absorbed electrons
Cons of transmission electron microscopes:
- specimens require lots of preparation + must be very thin
- specimens cannot be living- must be observed in a vacuum
- preparation techniques can introduce artefacts- features that are not normally part of the sample
- expensive + require training to use
Principles of scanning electron microscopes:
- provide 3D images
- high magnification
- great depth of field
- provide a detailed surface view of surface structures
Cons of an SEM:
- cannot view live specimens
- have to coat specimens with a small layer of metal
- resolution worse than TEM
- expensive + require training
What is magnification?
This refers to how many times larger an object appears compared to its actual size. It does not necessarily improve the detail of the image, just the size.
What is resolution?
This is the ability to distinguish between two points that are close together as separate points. Higher resolution provides more detail and clarity in the image.
Principles of cell fractionation:
- homogenisation
- buffer solution
Homogenisation:
Cells are broken up using a blender or homogeniser to release organelles. The resulting mixture, called homogenate, is filtered to remove unbroken cells and large debris.
Homogenisation:
Cells are broken up using a blender or homogeniser to release organelles. The resulting mixture, called homogenate, is filtered to remove unbroken cells and large debris.
Buffer solution:
The homogenate is kept in a cold, isotonic, and buffered solution to prevent damage to organelles. Cold reduces enzyme activity, isotonic prevents osmotic lysis, and buffering maintains the pH.
Parts of ultracentrifugation:
- centrifugation
- supernatant removal
Centrifugation:
The homogenate is placed in a centrifuge and spun at low speed. The heaviest organelles, like nuclei, sediment at the bottom.
Supernatant removal:
The supernatant is transferred to a new tube and spun at higher speeds. Successively lighter organelles, such as mitochondria and then ribosomes, sediment in subsequent spins.
