2.1.1 - Microscopes Flashcards
LM( How it works)
- A beam of light passes through the sample.
- The objective lens first magnify the image, then the eyepiece lens magnifies it again
LM (What it’s used for)
- Used to view living cells and tissues
- It can also be used to view cellular events in real time
LM (+)
- Cheap
- Can be used to observe living and dead cells
- Coloured images
- Doesn’t use harsh chemicals
- Small and portable
LM (-)
- Low magnification(x2000)
- Low resolution(200nm)
TEM (How it works)
- Electron beams pass through the specimen, the beam is focused to produce an image.
TEM (What it’s used for)
- Produce the most magnified images.
- To view sub cellular structures
TEM (+)
- High magnification(over x500000)
- High resolution(0.5nm)
- Can observe subcellular structures
TEM (-)
- Expensive
- Can only be used to observe dead cells
- Harsh chemicals used in prep can cause artefacts
- Large and difficult to move
SEM (How it works)
- A beam of electrons hit the surface of the specimen and bounce off, the electrons then produce an image
SEM (What it’s used for)
- To produce 3-D images of this surface of a specimen.
SEM (+)
- High magnification (x500000)
- High resolution (3-10nm)
- 3D images produced
SEM (-)
- Expensive
- Dead cells
- Harsh chemicals used can cause artefacts
- Black and white
- Large and difficult to move
Laser scanning confocal microscope
A laser moves across the specimen. Light is absorbed by fluorescent chemicals and radiated back from the specimen. Light from the specimen is filtered, it passes through the pinhole aperture. Only light from close to the focal plane is detected. Unwanted radiation does not go through the pinhole
LSCM
- View living cells and tissue
- Optical slicing through tissue
- Can see different layers
LSCM (+)
- View living cells in colour
- Sections can be made through the cell horizontally and vertically
- Higher resolution than light microscope
LSCM (-)
- Mag and res lower than electron microscope
- More complex than light microscope
Magnification
- Magnification is how many times larger the image is than the actual size of the object being viewed
Resolution
- Resolution is the ability to see individual points as separate entities
- It is the amount of detail seen
Sample preparation
- Dry mount is where solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning. The specimen is placed on the centre of the slide and a coverslip is placed over the sample.
- Wet mount is when specimens are suspended in a liquid such as water or an immersion oil. A coverslip is placed on from an angle.
- Squash slide is when a wet mount is first prepared then a lens tissue is used to gently press down the coverslip then the sample is squashed between two microscope slides.
- Smear slide is when the edge of a slide is used to smear the sample, creating a thin even coating on another slide. A coverslip is then placed over the sample.
Preparing slides
- Fixing : Chemicals like formaldehyde are used to preserve specimens in as near natural state as possible.
– Sectioning: specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. This can then be sliced thinly with a microtome. - Staining : Specimens are often treated with multiple stains to show different features
- Mounting : The specimens are then secured to a microscope slide any coverslip is placed on top
Uses of staining
- To highlight organelles
- Some cells are colourless
- To increase contrast
Differential staining
It can be used to distinguish between two types of organisms that would otherwise be hard to identify.
Eyepiece graticule and stage micrometer
- Put the stage micrometer in place and the eyepiece graticule in the eyepiece
- Get the scale on the micrometer slide in clear focus
- Align the micrometer scale with the eyepiece graticule scale
- Measure the size between each division using the stage micrometer
• The eyepiece graticule has to be calibrated for each objective lens separately because the size of the divisions change.
• The eyepiece graticule has no units because the size of the division changes when the magnification changes. So it has to be calibrated for each objective lens and a micrometer is used to measure the size of each division.
Why can’t small organelles be seen through a light microscope
- Too small
- Resolution of light microscope not high enough
- Wavelength of light too long
Magnification
A=I/M
M = Eyepiece x Objective
