Methods of studying cells Flashcards
Microscopes
instruments that produce a magnified image of an object
there are 2 types
* Light microscopes
* Electron microscopes
Magnification
how many times bigger the image is when compared to the object
* object= material put under the microscope
* image= appearance of the material when viewd under the image
* EQUATION:
mag = size of image/ size of real object
Resolution
resolving power is the minimum distance apart that 2 objects can be, in order to appear as seperate items
* greater resolution = greater clarity
Cell fractionation
process where cells are broken up and the different organelles they contain are seperated out
* isolates different organelles so they can be studied
* enables individual organelles structure and functions to be studied
1. Cells are broken open to release organelles and they are then seperated
2. Cells are prepared in a cold, buffered and isotonic solution
3. Homogenation
4. Ultracentrifugation
How are cells prepared for cell fractionation
cells are placed in a:
1. Cold solution- to reduce enzyme activity that may break down/damage the organelles
2. Isotonic solution- so its the same water potential as the tissue to prevent osmosis so organelles dont shrivel or burst
3. Buffered solution- so pH doesnt fluctuate to prevent damage to organelle structure and reduce the affect of functioning of enzymes
Homogenation
Cells are broken up, using a blender (homegeniser) and filtered
* this releases the organelles from cell
* resultant fluid, (homogenate), is filtered to remove any complete cells or large debris
Ultracentrifugation
filtered solution is spun at different speeds in a centrifuge. organelles are seperated by densities
* tube of filtrate is placed in the centrifuge and spun at a slow speed
* heaviest organelles, nuclei, are forced at the bottom of the tube where they form a pellet
* Fluid at the top of the tube (supernatant) is removed, leaving the pellet of organelles (nuclei)
* Supernatant is spun faster at a higher speed to remove the next pellet
* Repeats again until organelle required is removed
* Heaviest pellet: Nucleus, chloroplsts, mitochondria, lysosmes, ER and ribosomes
Light microscope
- used to view living/dead organisms
- view thin sections of larger plants + animals
- uses light waves
- cannot give a detailed information about organelles
- need to stain the specimen using chemical
- RESOLUTION= 0.2 micro m
- MAGNIFICATION= x1,500
Compare light microcopes to electron microscopes
- Light microcopes have a poor resolution
- Light microscopes have a magnification x1500 but electron
- Light microscopes have a long light wavelength, but electron microscopes have a very short wavelength
- Electrons are absorbed or deflected in air so a near-vacuum has to be created
TEM
Transmission electron microscope
* electron gun produces electron beam that passes through thin section on specimen
* Dense parts that absorb more electrons are dark, less denser parts alow beam to pass through so are light
* Image produced is a photomicrograph
* RESOLUTION= 0.1nm
* MAG= 50,000x
- samples are placed in a vacuum so living specimens cannot be observed
-complex staining process is required and image is not in colour
-specimen must be extremely thin
- image may contain artefacts- things that result from specimen preparartion- that may appear on the photomicrograph
- only creates a 2D- images
SEM
Scanning electron microscope
* directs a beam of electrons on to the surface of the specimen
* beam is passed back and forth and electrons are scattered by the specimess air molecules
* creates a 3D image
* Mag= x20,000
* RESOLUTION= 20nm
- samples are placed in a vacuum so living specimens cannot be observed
- preparing samples requires a high degree of skill and training due to complex staining
- image may contain artefacts- things that result from specimen preparartion
Eyepiece graticule
a glass disc that is placed in the eyepiece of a microscope
* A scale is etched on the disc
* scale= 10mm long, divided into 100 subdivisions
* scale is visible when you look down the eyepiece of the microscope
Calibration of eyepiece graticule
- A stage micrometre is used to calibrate the eyepiece graticule
1. line up stage micrometre and eyepiece graticule whilst looking through eyepiece
2. Count how many divisions of the eyepice graticule fit into 1 division on the micrometre scale
3. Each division on the micrometre scale is 10micro m and this can be used to calculate what 1 division on the eyepiece is at that current magnification - Actual size of the structures can be measured using the eyepiece graticule scale
Stage micrometre
used to calibrate the eyepiece graticule
* glass microscope slide with a scale etched on it
* typically 2mm long + subdivisions 10 micro m apart
