2.1.1c Microscopy Flashcards
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 (seeing two structures that are very close as two separate objects)
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
