2.1 - Microscopy Flashcards
What is Magnification?
Factor by which the image is larger than the actual specimen.
Magnification = size of the image = size of the real object
What is resolution? What is it determined by?
The minimum distance between two objects in which they can still be viewed as separate objects
Higher resolution = clearer image.
This is determined by the wavelength of light in a light microscope and by the wavelength of the electron beam in an electron microscope.
What is the formula to calculate magnification?
Magnification = Image size ÷ object size
What is the eyepiece graticule?
A small piece of glass with a measurement scale etched on its surface that fits inside a microscope eyepiece.
What is the stage micrometer? What is each division equal to? What is used for?
A microscope slide that has a ruler/scaled bar etched into it.
The scale is usually 1mm long and has 100 divisions so each division in 10µm.
It is used to calibrate the eyepiece graticule.
How do you calibrate the eyepiece graticule?
- Line up the stage micrometer and eyepiece graticule whilst looking through the eyepiece.
- Count how many divisions on the eyepiece graticule fit into one division on the micrometer scale.
- Each division on the micrometer is 10µm, so this can be used to calculate what one division on the eyepiece graticule is at that current magnification.
What are the 6 key features of scientific drawings?
- Draw in Pencil.
- Title the diagram to indicate what the specimen is.
- State the magnification that you are drawing it from.
- Annotate cell components, cells and sections of tissue visible.
- Do no sketch - only use solid lines that do not overlap.
- Do not colour in or shade.
What is differential staining?
A techniques involving many differential chemical stains being used to stain different parts of a cell in different colours.
E.g., crystal violet and acetic orcein.
What are the 2 types of microscopes? Give examples.
- Light (Optical) Microscope.
- Electron Microscope:
A) Transmission Electron Microscope (TEM)
B) Scanning Electron Microscope (SEM)
What are the advantages of using a light microscope? (5)
- Whole cells and tissues can be seen
- Easy sample preparation
- Cheap to buy (<£1k)
- Can use live specimens.
- Coloured image
What are the disadvantages of using a light microscope? (3)
- Low magnification (x1500)
- Low resolution (0.2µm)
- Specimens are thin = may not be representative + cannot see all organelles.
Describe how a transmission electron microscope (TEM) works
- The sample must be thin and stained.
- Pass a high-energy beam of electrons through a thin slice of specimen.
- More dense structures appear darker since they absorb more electrons.
- Focus image onto fluorescent screen or photographic plate using electromagnets in a vacuum.
Describe the advantages of using a Transmission Electron Microscope. (3)
- High magnification (x1,500,000)
- High resolution (0.5nm)
- Provides detailed images of interior substances.
Describe the disadvantages of using a Transmission Electron Microscope. (6)
- Can only see dead specimen
- Harsh chemicals used in preparation which can cause artefacts - complex staining method
- Expensive
- Training required + Vacuum required.
- Black and white images (false colouring).
- Extremely thin specimens required.
What can you see with a TEM?
2D images of details within organelles.
For example:
* cristae in the mitochondria
* grana in the chloroplasts
Describe how a scanning electron microscope (SEM) works.
- Focus a beam of electrons onto a specimen’s surface using electromagnets.
2, Reflected electrons hit a collecting device and are amplified to produce an image on a photographic plate.
Describe the advantages of using a Scanning Electron Microscope (SEM). (4)
- High magnification (x1,500,000)
- High resolution (5nm)
- Can see details of the surfaces of structures
- Produces a 3D image.
Describe the disadvantages of using a Scanning Electron Microscope (SEM). (6)
- Can only see dead material
- Harsh chemicals used in preparation which can cause artefacts - complex staining methods.
- Expensive
- Black and white image (false colouring).
- Thick sample is unusable - sample has to be very thin.
- Vacuum required.
What can you see with a SEM?
3D image of the surface of cells and organelles.
Describe how a laser scanning confocal microscope works.
The image is created as teh microscope scans the specimen point-by-point using a focused laser beam.
Can see a 2D or 3D image.
What are the advantages of using a laser scanning confocal microscope? (4)
- Can see living cells.
- Can observe cell processes by tracking molecules.
- Higher resolution than light microscopes (0.02µm).
- Higher magnification (100,000x)
What are the disadvantages of using a laser-scanning confocal microscope? (2)
- Cannot see deep into cell tissue as light cannot penetrate.
- More expensive and complex than a light microscope.
What is the benefit of having two lenses - objective & eyepiece?
- Objective lens magnifies the specimen
- Eyepiece lens magnifies image (from objective lens)
- Higher magnification (produced than with just one lens)
What is the magnification and resolution of a compound light microscope?
Magnification = x2000
Resolution = 200 nm
