microscopy Flashcards
describe how light microscopes work
uses light rays and a number of different lenses to produce an image viewed at the eyepiece
what are the two different types of microscopes
light microscopes
electron microscopes
what is the maximum resolution and magnification of a light microscope
resolution - 50-200nm
magnification - x1500
what are the two types of electron microscopes
transmission electron microscopes (TEMs)
scanning electron microscopes (SEMs)
laser scanning confocal microscope
describe how a transmission electron microscope work
an electron beam is directed at the sample with the electrons passing through less dense parts producing a contrasted 2D image
describe how a scanning electron microscope works
an electron beam is directed at the sample and reflected electrons are collected producing a 3D image of the cell surface
uses electro magnets
describe how a laser scanning confocal microscope work
specimen is treated with dye that is taken up by the cell components at different extents, a laser beam scans the specimen making the dye fluorescent and the emitted light is passed through a pinhole producing an image
a 3D image is produced and depth of image can be seen
image produced by a laser scanning confocal microscope
3D image with depth
pros of a light microscope
can observe living things
does not use harsh chemicals
easy to set up and use
inexpensive
can observe whole cells and tissues
cons of light microscopes
low magnification
low resolution - cant see organelles apart from the nucleus
cant observe Golgi, mitochondria, SER, RER
pros transmission microscopes
high magnification
high resolution
can see details inside the cells e.g. organelles
2D
cons of transmission microscopes
can only see dead material as materials must be put in a vacuum (all air molecules have been removed) because these will deflect the beams of electrons
samples must be stained with metals. these harsh chemicals used in preparation can cause artefacts
expensive
pros of scanning microscope
high magnification
high resolution
can see details of the surfaces of structures shown in 3D
cons of laser scanning microscopes
can only see dead material as materials must be put in a vacuum (all air molecules have been removed) because these will deflect the beams of electrons
samples must be stained with metals. these harsh chemicals used in preparation can cause artefacts
expensive
pros of laser scanning confocal microscopes
can see living cells
can observe cell processes by tracking molecules
flurescent tag
higher resolution than light microscope
depth of image can be seen
produces a 3D image
cons of a laser scanning confocal
more expensive than light microscope
more complex than light microscope
magnification and resolution of TEM
magnification: x500,000
resolution: 0.05 - 1nm
magnification and resolution of SEM
magnification: x500,000
resolution: 5-50 nm
define magnification
the number of times larger an image is than the actual object
resolution
the ability to distinguish separate points on an image as two separate object
magnification eq
magnification = image size / actual size
making an oinion cell slide
- carefully use a shape blade to peel a thin layer from the oinion so light can penetrate through and will enable a view of indibidual cells. Wear gloves to ensure there is no cross-contamination of foreign cells
- Gently place the sample onto the slide (using a wet mount) to prevent dehydration. (Wet mounts are used for mobile specimens/living tissues).
- stain the sample with a drop of iodine this will ensure you can seen and distinguish the internal parts of the cell more clearly/use more than more one stain to improve contrasts - place stain at edge of sample (no the centre) use blotting paper to remove excess stain - place stain on one stain
- gently lower a coverslip at a 45 degree angle to cover the smple to spread the cells. place at an angle to push any to the slide and reduce aur bubbles
what are the four slide preparation methods
dry mount
wet mount
squash slides
smear slides
dry mount
specimen is placed in the centre of the slide with cover slip
examples of dry mount
hair
pollen
dust
muscle or plant tissue
wet mount
specimen in suspended in a liquid such as water or immersion oil and a cover slip is placed from an angle
examples of wet mount
aquatic species
squash slides
wet mount is prepared first and a lens tissue is used to gently press the cover slip
examples of squash slides
root tips to observe cell division
examples of squash slides
root tips to observe cell division
smear slides
the edge of a slide is smeared with the sample, creating a thin and even smear with a cover slip over
examples of smear slides
blood cells
purpose of staining
to increase the contrast between different components making them more visible and identifiable
staining has to be….
air-dried before being heat-fixed by passing through a flame
methylene blue stain
staining living tissue - dark blue nucleus
light blue cytoplasm and bacteria cells
iodine solution stain
staining living plant cells
dark blue/black starch gains
acidified phloroglucinola
staining lignin red in the xylem walls
acetic orcein stain
stains nuclei and chromosomes red
eosin stain
stains (dead) cytoplasm and some organelles red
light green stain
stains plant cell walls green
preparing transmission electron microscope specimens
specimen needs to absorb electrons so it is treated with dye, heavy metal compounds are also used
why is a drop of water added when preparing a temporary mount
preparing cells being damaged by dehydration
why is an image unclear under a light microscope
use coarse focus for clearer image
specimen not thin enough for light to pass through
cross contamination with foreign cells
why are heavy metal compounds used as dye
absorb electrons well
method of making a blood smear
- pipette blood sample onto one side of microscope slide
- add fixant to make liquids not run off and add stain if you want to see a specific part of each cell
- add cover slip to spread the blood smear across the slide
- the cover slip must be added at an angle to prevent air bubbles
- leave to air dry
how to use a light microscope + calibration
- clip the slide onto the stage
- select the lowest powered object lens
- use the corse focus nob to move the objective lens to just above the slide
- look down the eyepiece and adjust the focus with the fine adjustment knob, until a clear image appears
- if a higher magnification is needed, swap to a higher powered objective lens and refocus - re-calibrate
to find the size of specimen: - fit an eyepiece graticule to the eyepiece
- place a stage micrometer (microscope slide with an accurate scale) on the stage - this will aid accuracy
- calibrate - identify the length in um of 1 epg
- measure the length of the specimen in epg
Explain how to measure the diameter of the nucleus of one of the white blood cells, when observing the cells through a light microscope. EQ
- use eyepiece graticules
- calibrate graticule using stage micrometer
- measure the diameter of the nucleus in graticules
- take repeated measurements and calculate a mean diameter
- use calibrate epg to calculate diameter
