3.2.1.3 Studying cells Flashcards
(21 cards)
What is magnification
How many times bigger the object appears
What is resolution
Ability to distinguish 2 points
Equation to calculate the ‘actual image size’
actual object size = image size / magnification
What measurements are used in the magnification equation
1000 micro m in 1 mm
1000 nm 1 micro m
How to measure an object using a microscope and gratitude
. Line up the gratitude with the object
. Count the no. Of eye piece units for the measurement
. Multiply the no. Of eye piece units by the calibration factor
. Calibrate the graticule using a stage micro m - how many micro m per eyes piece unit
How does an optical (light) microscope work
- light waves are focused onto an object and then onto the eye using glass lenses
Uses of an optical microscope
- study living and dead cells, cells can be stained in order to see features more easily
Limitations of the optical microscope
- relatively low magnification and resolution so cannot see all the organelles in a cell (only see the RER, lysosomes and ribosomes)
Resolution & magnification of the optical microscope
RESOLUTION:
0.2 micro m
MAGNIFICATION:
x1500
How does a transmission electron microscope (TEM) work
- beams of negatively charged electrons are focused using electromagnets and pass through the cell
- image appears darker the more electrons that are absorbed
Uses of the TEM
- high resolution so you can see the internal structure of organelles
Limitations of the TEM
. Vacuum required for microscope to function
. Specimen must be thin to allow transmission of elec.
. Staining with metals required
. Cells must be dead
. Artefacts may occur - features which aren’t part of the cell, but occur due to preparation methods
How does a scanning electron microscope work (SEM)
- beams of negatively charged electrons across the specimen
- knock off electrons from the specimen - gathered in the cathode ray tube from an image
Why are SEM’s good
- can show a 3D image
- used on thick specimens
Limitations of the SEM
. Vacuum required for microscope function
. Specimen must be thin in order to allow transmission of electrons
. Staining with metals required
. Cells must be dead
. Artefacts may occur
Resolution & magnitude for TEM
RESOLUTION:
0.1 nm
MAGNIFICATION:
X50,000,000
Resolution & magnitude for SEM
RESOLUTION:
20nm
MAGNIFICATION:
X1,000,000
2 processes used for extracting organelles
- Homogenation
- Ultracentrifugation
Process of homogenation
cells & organelles are kept in a solution which is:
. PH buffered - prevent denaturation of proteins which could affect enzyme function or damage organelles
. Cold - reduce enzyme activity, so organelles are not digested
. Isotonic - prevent H20 movement osmosing into or out of organelles & lysis of organelles
- Cells are broken up in a blender
- Fluid, homogenate is filtered to remove whole cells & large cell fragments
Process of ultracentrifugation
- tube of homogenate is placed in a centrifuge & spun at low speeds
. the heaviest organelles, nuclei, settle at the bottom of the tube - the remaining fluid, supernatent, is removed, placed in a new tube & spun at higher speeds
. Next heaviest organelles, mitochondria settle at the bottom of the tube - the process of removing the supernatent to a new tube & spinning at higher speeds is repeated several more times
. Heaviest remaining organelles settle each time
How to prepare a ‘temporary mount’ of a specimen on a slide
- Pipette a small drop of water onto a slide
- Use tweezers to place a thin section of your specimen onto the water drop
- Add a drop of stain - used to highlight objects in a cell
- Add the cover slip (stand the slip upright on the slide, next to water droplet, slowly tilt and lower it so it covers the specimen)
