1. Imaging in cell biology Flashcards
define resolution
the smallest structure you can see
what is the resolution of a light microscope
200 nm
what is the resolution of a TEM
0.1 nm
what is the resolution of a SEM
1 nm
what is a limitation of brightfield microscopy
samples must be preserved, sectioned and embedded. This process alters cell structure, so only gives a snapshot of dead cells
what is the purpose of ocular and objective lens’
magnify images
ocular lens = x10
objective lens and ocular couple to produce high mag.
what is the function of a microtome
sections specimens which have been preserved and embedded
what is a cryo-microtome
a microtome inside a freezer, can freeze tissues without using chemicals
why is a cryo-microtome good in immunolabelling
does not use chemicals to preserve tissues - only freezing
this does not alter epitope structure and so antibody binding is unaffected
why use a chemical stain (2 points)
- to see ultrastrucrure detail
- most cellular material is not capable of absorbing light
how do chemical stains work
bind to a class of molecules within a cell and absorb light, generating a visual image
give two examples of stains
haematoxylin and eosin
why do you fix tissues
prevents the autolysis
what is a common tissue fixative
formaldehyde
how thick are specimens generally sectioned
5 microns thick
how does fluorescent microscopy work
ultraviolet light excited fluorochrome in the dye, making it fluoresce
how does SYBR green work
binds to the groove in double stranded DNA, when a laser is shone on it, it emits lights = allows measure conc of ds DNA
advantages of fluorescent microscopy: 2
- cells may be fixed or living
- many fluorescent dyes can be used at once to label different cellular components
what are the limitations of fluorescent microscopy
the whole of the cell will fluoresce - creating a blurry image
what is immunolabelling used for
to identify and localise a particular antigen in a tissue
how do primary and secondary antibodies work
the primary antibody binds directly to the antigen
the secondary antibody is marked with fluorescent dye and binds to the primary antibody making them visible
how can we immunolabel the cytoskeleton
actin filaments can be stained with rhodamine-labelled phalloidin, this tag fluoresces red
microtubules can be stained with a tag that fluoresces green
what is the advantage of confocal microscopy
facilitates optical sectioning - we get fluorescence from only a section of the cell
produces a sharp image
what is a limitation of confocal microscopy
very expensive