Methods of Studying Cells

magnification =

image size / actual size

Light microscopes

• uses pair of convex glass lenses

• resolution of 2um (wavelength of light restricts resolution)

• EM = 1um

resolution

minimum distance apart that two objects can be distinguished as separate objects within an image

-greater resolution = clearer image

electron microscopes

-use beam of electrons focused by electromagnets inside vacuum

vacuum inside EM

stops particles in air deflecting electrons out of beam alignment

TEM

beam of electrons passes through thin section of specimen

-areas that absorb electrons appear darker on electron micrograph

SEM

beam of electrons passes across surface and scatters

-scattering pattern builds 3D image (dependant on specimen contours)

limitations of EM

-whole system must be in vacuum, live specimens cannot be observed

-complex staining process required, may introduce artefacts on image

-specimens must be very thin, so electrons can pass through

-SEM has lower resolving power than TEM

cell fractionation

process by which different parts and organelles of a cell are separated for study in detail

homogenation 1 

cells blended in homogeniser, forming resultant fluid of homogenate, which is placed in a centrifuge and spun at low speed

homogenation 2

heaviest organelles, nuclei, forced to bottom of tube, where thin sediment/pellet forms

homogenation 3

fluid at top (supernatant) is removed, leaves sediment of nuclei

supernatant transferred to another tube, spun at faster speed

pellet forms, containing next heaviest organelle, mitochondria

homogenation 4

speed increases each repeat, next heaviest organelle sedimented and separated out

buffer solution homogenate placed in is:

cold - inactivates any enzymes from breaking down organelles

same water potential - prevent organelles bursting under osmotic pressure

buffered - pH does not fluctuate