2.1.1 microscopy Flashcards
cell theory
- plant and animal tissues are made up of cells
-cells are the basic unit of all life - cells only develop from existing cells
magnification
how many times bigger an image appears, compared to the real
specimen
resolution
the ability of a microscope to distinguish between 2 points that are
close together. The higher the resolution, the clearer the image,
the more detail can be seen
what is a high resolution?
a clearer image can be seen, meaning there is more detail seen too
how many lenses does a compound light microscope have
2
name the 2 lenses a light microscope has
objective lens
eyepiece lens
what does the objective lens do?
it is placed near the specimen and produces a magnified image
what does the eyepiece lens do?
it is what the specimen is viewed through and it magnifies the image produced by the objective lens
what does the objective/eyepiece configuration in a light microscope allow for?
- higher magnification
- reduced chromatic aberration than in a simple light microscope
how is illumination provided in light microscopes?
by a light underneath the sample
how can you calculate the total magnification in a light microscope?
total = eyepiece mag x objective mag
how do you prepare a microscope slide?
- use a temporary mount (specimen is suspended in liquid)
1. pipette small amount of water onto slide
2. use tweezers to place thin section onto water
3. stain added to the sample
4. add cover slip, without getting any air bubbles in there
why do you stain the sample?
- To give CONTRAST to the image as some parts of the sample will take up
the stain and other parts won’t - Makes the image VISIBLE (without a stain, most biological structures
are transparent, will all look the same)
safety procedures whilst dealing with slide?
-carry a microscope with both hands
-store microscopes with the lowest power lens in place
-cover microscope with dust sheet when not in use
fixing
Chemicals (e.g. formaldehyde) used to preserve the specimen in as near natural state as possible
sectioning
specimen is dehydrated with alcohol, then placed in a wax mould to harden it, then sliced very thinly (thin enough to let light through)
staining
Provides contrast as diff parts of the specimen take up diff stains,
giving diff colours, which you can then see
mounting
specimens secured to a microscope slide, with a permanent cover-slip on top
RULES FOR DRAWING WHAT YOU OBSERVE WITH A LIGHT MICROSCOPE
- title
- state mag
- sharp pencil
- use as much paper as possible
- smooth, continuous lines
- do not shade
- clearly defined structures
- correct proportions
- label lines should not cross or have arrow heads
- label lines should be parallel to top of page
laser scanning confocal microscope
- uses laser beams to scan specimen, which can be tagged with a fluorescent dye, so fluorescent gives off light
- light is focused through a pinhole onto a detector, which generates a 3D image
use of pinhole in LSCM
means out of focus light is blocked, so microscope can produce clearer image than normal light microscope, increasing resolution
what are LSCMs used for?
thick specimens
what does the laser in an LSCM do?
improves illumination as it gives a higher light intensity
what does it mean by LSCM have depth selectivity?
they can focus on structures
at different depths within a specimen
what is depth selectivity useful for?
observing whole living
specimens as well as cells
define fluorescence
emission of light that has been absorbed
limitation of LSCM that cannot be used for deep tissue imaging?
penetration of sample is limited
cm to mm
x10
mm to μm (micrometres)
x1000
μm to nm
x1000
what happens if resolution is poor?
two objects that are very close together will be seen as one
why do light microscopes not have good resolution? (4 points)
- wavelength of light is long.
- Light waves get reflected from structures in the specimen as they pass through it. These waves then get diffracted (spread out) so that they end up overlapping with each other.
- this means structures from where this light came are no longer seen
as being separate to each other and the detail is lost. - the best resolution is 200nm
- this also affects the mag
how can you calibrate a microscope so the specimen can be measured?
- eyepiece graticule has a scale, with no units
- stage micrometer is viewed
- line them up
- find out what each EPG division’s unit is
if you want to look at the specimen using the different objective lenses what do you have to do and why?
re-calibrate as on each magnification , the proportion of the stage micrometer visible changes
how does a transmission electron microscope work?
- a beam of electrons is fired at the specimen
- a series of electromagnets focuses the electrons on to the specimen
- denser parts of the specimen absorb electrons (stop them passing through) and less dense areas let the electrons through
- the electrons are focused on to a screen which detects them, and an image is produced.
type of image produced by a TEM
2D, black and white, shows internal detail of specimen
why is a beam of electrons being used better than light?
the wavelength of light is shorter than of the beam, so you get a higher higher resolution and mag in a electron microscope
limitations of a TEM and SEM
- specimens need to be sliced very thinly to allow electrons through
- needs to be a vacuum inside as any air molecules would deflect the electron beam
- so only dead specimens can be viewed
The specimen sits on a copper grid rather than a glass slide, why?
- A glass slide would stop the flow of electrons
- The electrons would not reach the screen
- Copper grid has holes in it to let electrons through
how does a scanning electron microscope work?
scan a beam of electrons across the surface of a specimen, and any
reflected electrons are collected.
what type of image is produced by a SEM?
3D surface image of the specimen
The advantage of the SEM compared to TEM
the specimens do not need to be cut
quite as thin, since the electrons do not need to pass through
disadvantage of the SEM compares to the TEM
the images are lower
magnification and resolution
staining in electron microscopy
solutions of heavy metal salts that attach to some parts of the specimen and not others. heavy metal ions scatter the electrons that are fired at the sample, giving contrast between structures.
9 differences between light and electron microscopes
Light
1. inexpensive to buy
2. small and portable
3. simple sample preparation
4. vacuum not required
5. can view living or dead samples
6. natural colour can be seen
7. coloured stains/dyes used for contrast
8. Max. magnification X2000
9. Max resolution 200nm (0.2µm)
Electron
1. expensive
2. large, needs to be installed
3. complex sample prep
4. needs vacuum inside
5. can only view dead sample
6. black and white images
7. Heavy metal salts used for contrast
(“false colour” can be added digitally)
8. Max magnification;
X 1000 000 for TEM
X 500 000 for SEM
9. Max resolution;
0.5nm for TEM
3-10nm for SEM
max mag of light microscope
x2000
max resolution of light microscope
200nm (0.2µm)
max mag of TEM
x 1,000,000
max mag of SEM
x500,000
max resolution of TEM
0.5nm
max resolution of SEM
3-10nm
what are artefacts?
These are visible structures on the image which are not really part of the specimen;
e.g. air bubble when preparing a slide for a light microscope
specks of dust when preparing specimens for an electron microscope
where are artefacts most likely to occur?
electron microscope because the sample preparation is a lot more complicated
