Microscopes Flashcards
what are microscopes?
instruments that produce an magnified image of an object
what is magnification?
the degree to which an image increases in size from its original image and size
what is resolution?
the ability to distinguish between two points
formula for magnification
actual size
formula for actual size
magnification
to convert millimetre to nanometre …
x1000
to convert nanometre to centimetre …
/10,000
what is cell fractionation?
process by which cells are broken up and organelles are separated out
what type of solution is the tissue placed in before fractionation?
cold, buffered solution with same water potential
why is the solution cold?
to reduce enzyme activity that might break up cells
why is the solution buffered?
to prevent changes in pH so that structure/function of enzymes is not altered
why is the solution isotonic?
to prevent organelles shrinking/bursting by osmosis
how are the cells broken up?
in a homogeniser to release organelles
why is the resultant fluid filtered?
to remove debris and complete cells
what happens during ultracentrifugation?
organelles in filtered homogenate are separated out in a centrifuge
how does a centrifuge work?
- spins homogenate at low speed for 10 mins - heaviest organelles, nuclei, forced to bottom forming a sediment
- supernatant removed leaving sediment of nuclei
- supernatant spun again at faster speed - removes mitochondria
- process continues increasing speed each time
how to prepare a slide
- add drop of water onto slide (temporary mount) using pipette
- use tweezers to place specimen on top of water drop
- add drop of stain
- add coverslip by standing upright next to drop and carefully lowering onto drop without creating air bubbles
what are the disadvantages of preparation of specimen for microscopy?
due to complex process:
- artefacts can form (e.g. air bubbles, fingerprints)
- resolution affected
name 5 types of stain
- methylene blue
- eosin
- iodine
- sudan red
- acetic orcein
what is methylene blue used for?
makes nuclei more visible
what is eosin used for?
stains red blood cells and cytoplasmic materials pink
what is iodine used for?
stains cellulose yellow
what is sudan red used for?
stains lipids
what is acetic orcein used for?
stains chromosomes red
name two advantages of using electron microscopes
- electrons have shorter wavelength than light so higher resolving power
- electrons have negative charge so can be focusing using electromagnets.
why do electron microscopes have vacuum inside?
prevent electrons colliding with air molecules
name two types of microscopes
- transmission electron microscope
- scanning electron microscope
describe how a transmission electron microscope works
- electron gun fires beam of electrons which are focused by condenser magnet onto specimen
- specimen absorbs some electrons which makes it dark and lets some electrons pass through making it light
- 2D image projected onto fluorescent screen producing a photomicrograph
describe how a scanning electron microscope works
- electron gun fires beam of electrons which is directed onto surface of specimen
- beam is passed back and forth across portion of specimen in pattern
- electrons are scattered and pattern of electrons depends on contour of specimen’s surface
- 3D image formed by computer analysis of pattern of scattered electrons
resolution of TEM
0.1 nm
resolution of SEM
20nm
maximum magnification of TEM
x 500,000
maximum magnification of SEM
x 100,000
limitations of TEM
- artefacts
- vacuum
- thin specimen
- 2D
- staining
- black and white
- expensive
limitations of SEM
- artefacts
- vacuum
- staining
- black and white
- expensive
- lower resolution than TEM
advantages of light microscopes
- specimen can be alive
- inexpensive
limitations of light microscopes
- poor magnification/resolution
