microscopy Flashcards
What are the four types of sample preparation for light microscopes?
- Dry mount
- Wet mount
- Smear slide
- Squash slide
What is a dry mount?
When thin slices or whole specimens are viewed, with just a coverslip placed on top.
What is a wet mount?
- When the specimens are added to water or a stain before the coverslip is lowered on with a mounted needle.
- can be used to observe aquatic organims
What is a smear slide?
- a smear slide is created by placing a drop of the sample at one end of the slide and using the edge of another slide to smear it across.
- can be used to view blood cells
what is squash slide?
- wet mounts, but you also push down on the coverslip to squash the sample.
- this is so it is thin enough for light to pass through.
- is used for viewing chromosomes in mitosis.
how do electron microscopes work? what can they produce? (generally)
Electron microscopes:
- The image is created using an electromagent to focus the bean of negatively charged electrons
- the beam of electrons has a very short wavelength- so a high resolution.
- EM must be in a vacuum so only non-living species can be examined
- the image is black and white as the sample must be stained.
how does a TEM work?
TEM:
- Extremely thin specimen stained and put in vaccum.
- Electron gun produces a beam of electrons that will pass through the specimen.
- some parts of the specimen absorb the electrons and this makes them appear darker
- produces a 2D image where you are able to see the internal structure of cells.
how does a SEM work?
SEM:
- Specimen doesnt need to be thin
- Electrons are beamed onto the surface and the electrons are scattered in different ways, depending on the contours of the specimen.
- produces a 3D image of the surface of the specimen.
how does a laser scanning confocal microscope work?
Laser scanning confocal:
- type of flourescent microscope
- uses a high light intensity to illuminate the specimin in a flourescent dye
- the microscope scans the specimen point by point using a focused laser beam to create a 2D or 3D image.
- can produce a 3D image where you can also see tiny structures that would be hard to section off
what is the resolution of a light, scanning electron, and transmission electron microscope
- LM- 200 nm
- SEM- 3-10 nm
- TEM- 0.2-0.5 nm
what is the magnification of a light, scanning electron and transmission electron microscope?
- LM- 1500-2000 x
- SEM- 100,000 to 500,000 x
- TEM- 500,000 to 2 million x
what are the rules for scientific drawings?
- draw in pencil.
- title of the diagram has to indicate what the specimen is.
- must state the magnification
- must label the key features
- must annotate cell components, cells, and sections of the tissue visible. This must state what colour and shapes they are and not their functions.
- only use solid lines that do not overlap
- no colouring or shading
- label lines must be horizontal, drawn with a pencil and ruler and not have arrowheads on them.
what is the gram staining technique?
- is used to separate bacteria into 2 groups
> gram- positive bacteria and gram- negative bacteria
PROCESS
- crystal violet is applied to the specimen, then iodine to fix it in place
- the slide is washed with alchohol
- gram-negative bacteria have thin walls and will lose the stain. These will then be stained with safranin dye (a counterstain)
- this makes gram-negative bacteria appear red.
- gram- positive bacteria will appear blue or violet.
what are the positively charged dyes and how do they work?
- crystal violet
- methylene blue
- they are attracted to negatively charged materials in the cytoplasm
what are the negatively charged dyes and how do they work?
- nigrosin
- congo red
- they are repelled by negatively charged cytosol
- the dyes therefore stay outside the cell so the cell interior contrasts with the background
what are the 4 stages of preparing a slide?
1) fixing - chemicals are used to preserve specimins in as natural state as possible.
2) sectioning - specimens are dehydrated, placed in a mould of resin/wax, then sliced thinly with a knife.
3) staining - the specimen is treated with stains to show structures.
4) mounting - the specimen is secured on the slid with a coverslip.
(remember- farting sloths seem mythical)
what are the disadvantages of electron microscopes?
- expensive
- must be trained
- can only be used in controlled environments and spaces
- preparation is very complex and can produce artefacts
- samples must be dead for the vacuum
what is the light and electron microscope’s way of focusing?
- light: glass lenses
- electron: electromagnets
why do electron microscopes use vacuums?
because the scattering of electrons by air molecules can interfere with image production
what are the requirements for specimens for light and electron mciroscopes?
light:
- can be alive or dead
- can be stained
- must be relatively thin
electron:
- always dead
- in TEM specimens must be extremely thin
- in SEM specimens are not required to be thin
how are light and electron microscopes stained?
light:
- coloured dyes
Electron:
- heavy metal ions which will scatter electrons
The image appears:
- 2 dimensional with a light background
- you can see a wide variety of colours
- you can only see details such as nuclei and large vacuoles
what microscope produced this image?
Light microscope
The image appears:
- greyscale or in false colour
- 3d image of the surface or inside of cells
- outside surface seen in great detail
- black background
what microscope produced this?
Scanning electron microscope, SEM
The image appears:
- greyscale or with false colour
- 2d
- small structures may appear grainy
- pale background
- objects closer than 200m are separate
what microscope produced this image?
Transmission electron microscope, TEM
