Microscopes Flashcards
What are the principles of a light microscope ?
-long wavelength light shone on specimen
What can you see with a light microscope?
Nucleus
Nucleolus
Cell membrane
Cytoplasm
Advantages of a light microscope
-colour images can use living samples
Disadvantages of a light microscope
-lower resolution due to long wavelength of light
lower magnification
-smaller organelles in cells are not visible
-max magnification is x1500
-max resolution is 0.2 micrometers
What are the principles of a scanning electron microscope?
-scans electrons across surface of objects
-knocks of electrons which are gathered by cathode to form
What can you see with a scanning electron microscope?
Surface of a specimen eg shape of cell
Advantages of an SEP
Higher magnification
Higher resolution
Details on image (texture and 3d depth )
Thick specimens
Disadvantages of an SEP
[samples must be in a vacuum ]
Lower resolution than TEM(20nm)
What are the principles of a transmission electron microscope?
Beam of electrons focused by electromagnet
Some parts absorb elements and appear dark
Extremely thin specimens
What can you se with a TEM?
Chloroplasts,mitochondria ,Golgi, E.R, ribosomes ,lysosomes, cell wall
Advantages of a TEM
Higher resolution than light microscope due to shorter wavelength of electron beam
Disadvantages of TEM
Need a vacuum so can’t view living specimens
Complex staining process
Black and white image
Very thin specimens needed
Image contains artefact
Define magnification
How many time larger rhe image is compared to the object
Define resolution
Minimum distance between two objects in which they can still be wirewed as separate
How many millimetres in a metre?
0.001
10^-3
How many micrometers in a metre?
0.000001
10^-6
How many nanometres are in a metre ?
0.000000001
10^-9
Magnification calculation
Image/actual
Resolution define resolution
The resolution or resolve in power of microscope is a minimum distance apart that two objects can be in order for them to appear as separate items
What is necessary to study the structure and function of various organelles?
In order to study the structure and functions of the various organelles make up cells is necessary to obtain large numbers of isolated organelles
Define cell fractionation
The process where cells are broken up the different organelles they contain are separated out
What is the tissue placed in before cell fractionation can begin ?
Before cell fractionation can begin the tissue is placed in a cold buffered solution of the same water potential as the tissue
Describe the solution for cell fractionation
-cold-to reduce enzyme activity that might break down the organelles
-is of the same water potential as the tissue – to prevent organise bursting or shrinking as a result of osmotic gain or loss of water
-buffered-so that the pH does not fluctuate. Any changed in pH could alter the structure of the organelles or affect the functioning of enzymes
What are the two stages of cell fractionation?
Homogenation
Ultracentrifugation
Describe homogenation
Cells are broken up by a homogeniser
Releases the organelles from the cell
Resultant fluid known as homeogenate is then filtered to remove any large piece of debris
Describe ultracentrifugation
It’s the process by which the fragments in the filtered homegenate are separate in a machine called a centrifuge
This spins tubes of homeogenate at very high speed in order to create a centrifugal force
What is the ultracentrifugation process for animal cells.
Tube of filtrate is placed in the centrifuge and spin at slow speed
Heaviest organelles, the nuclei are forced to the bottom of the tube where they form a thin sediment or pellet
Fluid at the top of the tube(supernatar) is removed leaving just sediment of nuclei
The supernatar is transferred to another tube and spun in the centrifuge at a faster than before
The next heaviest organelles the mitochondria are forced to the bottom of the tube
The process is continued in this way so that at each increase in speed the next heaviest organelle is sedimented and separated out
How do you measure the size of objects using a light microscope ?
Eyepiece graticule
Why can the scale in the eyepiece graticule not be used directly to measure the size of objects under a microscope?
Each objective lens will magnify to a different degree-the graticule must.first be calibrated for a particular objective lens -once calibrated in this way the graticule can remain in position for further use provided the same objective lens is used
What do you need to use to calibrate an eyepiece graticule ?
You need to use a special microscope slide called a stage ,micrometer
How do you calculate the scale for different kinds objective lenses?
Divide it by however many times bigger the magnification is
