microscopy Flashcards
define magnification
how much bigger the image is compared with the actual size of the object
what is the equation to calculate magnification
image size/ actual size
how many mm in a m
1m=1000m
how many um in a mm
1mm=1000um
how many um in a m
1m= 1000000um
how many um in a nm
1um=1000nm
define resolution
the smallest distance two points can be separated and still be seen as separate items
(exam q) why do we use ‘this’ unit
- it is the most appropriate unit
- avoids the use of excessive decimal places
- avoids the use of using unnecessary standard form
to what limit can the human eye can see
+- 0.25mm
what are the features of the light microscope (4)
- limited to a resolution of 0.4um
- can use living specimens
- easy to prepare specimen
- images produced in colour
what are the features of the electron microscope (6)
- has a resolution up to 0.1nm
- specimen must be dry, dead and extremely thin
- extremely complex preparation of the specimen
- images produced in black and white
- slicing the sample to make it thin can damage the inter-molecular structures
- image can produce artifacts (things on the object that arent actually real) e.g. air bubbles
describe the resolving power of the transmission electron microscope
it has a maximum resolution of 0.1nm which is not always achieved due to the difficult specimen preparation and the high energy (electron) beam which can destroy the specimen
advantages of the light microscope (3)
- can see living specimens
- easy to prepare specimen
- variety of coloured stains
disadvantages of the light microscope
- low resolution so organelle details/smaller components are not visible
advantages of the transmission electron microscope (2)
- has a very high resolution at high magnification
- can identify detailed organelle/sub-organelle structure
disadvantages of the transmission electron microscope (4)
- specimen not alive (in a vacuum)
- difficult preparation (very thin specimen, complex staining)
- produces a black and white image
- artefacts can spoil image
to what resolution can the scanning electron microscope reach to
20nm
advantages of the scanning electron microscope (1)
- 3D images show structural formation
disadvantages of the scanning electron microscope (3)
- specimens are not alive
- difficult to prepare specimen
- produces a black and white image
why do we stain specimens
it helps to identify specific features of cells and improves contrast within the specimen
name the three processes to fractionate a cell (isolating and separating individual components)
1- homgenisation
2- filtration
3- differential ultracentrifugation
describe the process of homogenisation
cells vibrated or grinded in a blender, breaking them open.
why is a cold isotonic buffer used in homogenisation
-cold to stop organelle activity (hydrolytic enzymes in lysosomes)
-> lysosomes used to digest material inside the cell. contain hydrolytic enzymes which hydrolyses the components in the homogenate (biological molecules) such as polypeptides
- isotonic to prevent the movement of water in and out of the cell through osmosis (prevents organelles bursting through the movement of water in)
why is a buffer solution used in homogenisation
it prevents change in PH levels, stopping important proteins from being denatured
describe filtration
solution is filtered through a gauze to remove cellular debris
describe differential ultracentrifugation
- solution spun in a centrifuge at a low speed
- heaviest organelles fall to the bottom (form a pellet), other organelles stay suspend in the fluid above the pellet (called the supernatant)
- pellet removed or supernatant depending on what you are looking for-
- (pellet removed) supernataant spun at a higher speed
- pellet forms. supernatant removed (these contain the lightest organelles)
