2.1.1c Microscopy Flashcards
(33 cards)
What is magnification
How many times bigger the image produced by the microscope is than the real life specimen
What is resolution
The ability to distinguish between 2 objects that are close together (ability to separate 2 objects/see more detail)
How detailed the image is
How to calculate magnification of a light microscope
Total magnification = Eyepiece lens magnification (10) x Objective lens magnification
Magnification equation
IAM triangle
Image size
Actual size Magnification
See images for labelled diagram of microscope
Description of Light microscope
- Use light to form an image
- Lower resolution than electron microscopes (max of 0.2pm) so usually used to view eukaryotic cells (sa nuclei & possibly mitochondria & chloroplasts)
- This is bc it is difficult to distinguish between (resolve) two objects that are closer than the wavelength of light (500-650nm)
- Maximum useful magnification is x1500
Description of Electron microscope
- Use electrons to form an image
- Higher resolution than light microscopes - so give more detail bc a beam of electrons has a much smaller wavelength than light.
- Maximum resolution of 0.2nm (1000x greater than optical microscopes) so is able to resolve 2 objects that are extremely close tg
- Can observe small organelles sa ribosomes, ER & lysosomes
- 2 types of e microscopes: TEM & SEM
Advantages of Light microscope
- inexpensive to buy & operate
- small & portable
- simple sample preparation
- vacuum is not required
- natural colour of sample is seen (or stains are used)
- specimens can be living or dead
Disadvantages of Electron microscope
- expensive to buy & operate
- large & needs to be installed
- complex sample preparation which often distorts material
- vacuum is required
- black & white images produced (can only be coloured digitally)
- specimens must be dead
Electron microscopes: What are Scanning electron microscopes
- SEMs scan a beam of electrons across the surface of the specimen. This bounces off the surface & knocks off electrons from the specimen, which are detected in a cathode ray tube forming an image.
- SEMs therefore form 3D images that show the surface of a specimen
- Magnification of x500,000 or less
Electron microscopes: Advantages of Scanning electron microscope
- Can be used on thick or 3D specimens
- allow external 3D structure to be observed
Electron microscopes: Disadvantages of Scanning electron microscope
- Lower resolution than TEMs
- Cannot be used on live specimen (unlike optical microscopes)
- Do not produce a colour image (unlike optical microscopes)
Electron microscopes: What are Transmission electron microscopes
- TEMs use electromagnets to focus a beam of electrons, which is transmitted through the specimen
- Denser parts of the specimen absorb more electrons, making them appear darker on the final image
Electron microscopes: Advantages of Transmission electron microscope
- Provide high resolution images, so great detail of a range of organelles
- Internal structures can be seen
- High magnification of x1,000,000 or more
Electron microscopes: Disadvantages of Transmission electron microscope
- Can only be used w very thin specimens.
- Cannot be used to observe live specimens bc of the vacuum inside the TEM. Plus all the water must be removed from the specimen
- Lengthy treatment required to prepare specimens. Artefacts could be introduced - these look like real structures but are acc the results of preserving & staining
- Do not produce a colour image & are in 2D
What are Laser scanning confocal microscopes
- relatively new tech
- cells are viewed and stained w fluorescent dyes
- a thick section of tissue or small living organisms are scanned w a laser beam (focused light)
- the laser beam is reflected by the fluorescent dyes
- multiple depths of tissue section/organisms are scanned to produce an image. As if the laser beam is building the image layer by layer
Advantages of Laser scanning confocal microscopes
- used on thick or 3D structures
- external 3D structure is observed
- very clear, high resolution bc the laser beam can be focused at a very specific depth. Cytoskeleton can be observed
Disadvantages of Laser scanning confocal microscopes
- slow process & takes a long time to obtain an image
- laser could cause photodamage to the cells
What is significant about tissues used in microscopy being transparent
Many tissues used in microscopy are transparent, letting light & electrons through them
- this makes it difficult to see details
- stains are often used to colour the tissues
Staining for light microscopy
For light microscope, coloured dyes are used
- The stain is taken up by some parts of the object more than others - the contrast makes the different parts show up
- Specimens absorb specific colours of light whilst reflecting others, making structures visible
- Diff stains are used to make diff things show up (eosin used for cytoplasms, methylene blue for DNA)
- More than 1 stain can be used at once
common stains: methylene blue and eosin
Staining for electron microscopy
For e microscopes, specimen must be stained to absorb/scatter the electrons
- electrons have no colour so dyes cause tissues to show up black/grey
- objects are dipped in solution of heavy metal compounds (like lead). The metal ions scatter the electrons, creating contrast - some parts of object shoe up darker than others
- colours are often added to image using image processing software
Converting units
nm
/1000
μm
/1000
mm
/10
cm
/100
m
/1000
km
What is the magnification of the eyepiece lens
x10
How to deal with scale bars (microscopes calc)
- measure actual length of scale bar w ruler (convert from cm to mm and then to μm)
- divide by length it represents
- this gives the magnification