3.2.1.3 - methods of studying cells Flashcards
how do you calculate magnification?
image size/actual size
what are the key unit conversions?
1m = 1000mm
1mm = 1000 um
1um = 1000nm
what are the key parts of a light microscope?
eyepiece, turret, objective lens, stage, coarse focus knob (moves stage up and down), fine focus knob (adjusts objective lens)
how do you set up a light microscope?
- prepare specimen - cut thin layer of onion cells, place on glass slide, add a small amount of iodine solution, place cover slip at an angle and let in drop on to avoid air bubbles, blot off excess iodine solution
- start observing using objective lens with lowest magnification
- use coarse focus to manually focus image
- switch to a high magnification and use the fine focus knob to focus
how do you calculate magnification using a scale bar?
- use a ruler to measure the length of the scale bar in mm
- use a ruler to measure the length of the given object in mm
- work out how much bigger the object is compared to the scale bar (divide length of object by length of scale bar)
- multiply the length represented by the scale bar by how much bigger the object is to get the actual length go the object
how many time can modern light microscopes magnify?
around 1000 times
what is the main advantage of light microscopes?
they can be used on living cells (can look at processes like cell division)
however sometimes a stain must be used which may kill the cell
what is the main disadvantage of light microscopes?
their resolution
what is resolution?
the ability to distinguish between two separate objects
what is the definition of resolution?
the minimum distance between two objects where they can still be seen as two separate objects
what is the limit of resolution for a standard light microscope?
around 200nm
why do light microscopes have a low resolution?
due to the nature of light because it has a wavelength of about 400nm-700nm
how does the resolution of an electron microscope compare to a light microscope?
it is around 2000 times better
how do electron microscopes work?
- an electron gun produces a beam of electrons
- the electrons pass down the microscope
- the inside of the microscope contains a vacuum so electrons pass through without bouncing off molecules in air
- electromagnets are used to focus the electron beam (electromagnetic lenses)
- the specimen is placed in the path of the electron beam and electrons pass through some parts more easily than others
- the final image is produced on a fluorescent screen
what is the limit of resolution for an electron microscope?
around 0.1nm
what are the general disadvantages of using an electron microscope?
the interior is a vacuum so living specimens can’t be viewed because all water must be removed from them
using one requires careful staining of the specimen, which must be very thin
it can lead to artefacts
what are the two types of electron microscope?
scanning electron microscopes (SEM) and transmission electron microscopes (TEM)
what is an artefact?
false images created by the staining process or conditions within the microscope
how do TEMs work?
an electromagnet is used to produce a beam of electrons which passes through the specimen
denser areas of the specimen appear darker because more electrons are absorbed
what are advantages of TEMS?
produce high resolution images so internal structures can be seen
what are disadvantages of TEMs?
only work if specimen is thinly sliced, produces 2D images, doesn’t produce colour image
how do SEMs work?
electrons are scattered from the surface of the specimen and detected
what are advantages of SEMs?
specimen doesn’t have to thinly sliced, produces 3D images
what are disadvantages of SEMs?
has a lower resolution than a TEM, specimen must be coated with metal which may lead to artefacts
what is an eyepiece graticule?
a scale on a glass disc found inside light microscopes
what does the eyepiece graticule do?
measure the size of objects being viewed under the microscope
what happens if you are viewing a specimen and change the objective lens?
this means you have changed the magnification, so you have to calibrate the eyepiece graticule to work out the size of the distance between each division at that particular magnification
what is a stage micrometer?
a glass slide with a scale on which you place on the stage and use to calibrate the eyepiece graticule
how are stage micrometers structured?
they are usually 2mm long and they have subdivisions which are 10um apart
how are eyepiece graticules usually structured?
they are 10mm long and each mm is divided into 10 parts - each small division is 0.1mm/100um
what is the method for using a stage micrometer to calibrate the eyepiece graticule?
- line up the stage micrometer and eyepiece graticule while looking through the eyepiece
- count how many divisions on the eyepiece graticule fit into one division on the stage micrometer scale
- each division on the stage micrometer is 10um, so use this to calculate what one division on the eyepiece graticule is at that magnification (length of stage micrometer division/ number of divisions on eyepiece graticule)
- measure the size of the cells or organelles using the divisions on the graticule and multiply the number of divisions by the length of each division
what is cell fractionation?
a method of breaking apart cells to obtain and study the organelles
what are the three stages involved in cell fractionation?
homogenisation, filtration and ultracentrifugation
what happens in the homogenisation stage?
the cells are blended in a homogeniser with a solution to produce a homogenate
what are the features of the solution in the homogenisation stage and why?
cold - reduces the activity of the enzymes that break down organelles
isotonic - stops water moving into cells via osmosis which would cause them to expand and become damaged
buffer - prevents organelle proteins, including enzymes, from becoming denatured
what happens in the filtration stage?
the sample is filtered using filter paper to remove any debris
what happens in the ultracentrifugation stage?
the filtrate is placed in a centrifuge and spun at a low speed, and the heaviest organelles (nuclei) are forced to the bottom of the tube and form a pellet
the fluid at the top (supernatant) is transferred to another tube and spun at a slightly faster speed to produce the next heaviest organelle (mitochondria)
the process continues so each time the speed increases the next heaviest organelle is separated (nucleus, mitochondria, lysosome, ribosome)
