2.1.1 Microscopy Flashcards
What are the 4 types of microscope?
• Light microscope
• transmission electron microscope [TEM]
• scanning electron microscope [SEM]
• laser scanning confocal microscope (type of light microscope)
How does a Scanning Electron microscope work?
A beam of elections is sent across the surface of the specimen and the reflected electrons are collected to form the image
Does a scanning electron microscope produce a 2D or 3D image?
SEM produces a 3D image
Does a SEM give a higher or lower resolution than a TEM
SEM produces lower resolution images than TEM
How does a Transmission electron microscope work?
Use electromagnets, a beam of electrons pass through the specimen and focussed to produce the image.
Denser parts of the specimen absorb more electrons, so they look darker.
Do transmission electron microscopes have a higher or lower resolution than SEM
Transmission electron microscopes have a very high resolution
How does a laser scanning confocal microscope work?
• Use laser beams,
• A single spot of light is moved across the sample (point illumination).
• Specimen is dyed with fluorescent dye
• Laser causes dyed parts to fluorescence - give off light
• light filtered through pinhole onto detector
• pinhole means out of focus light is blocked, producing a clear image.
Does a laser scanning confocal microscope produce a 3D or 2d image
Laser scanning confocal microscope images can be both 2D and 3D
Properties of light microscope:
Uses…
Magnification
Resolution
Dead / alive specimen
Light microscope:
Uses light radiation and glass lenses to focus
Magnification x1500 - x2000
Resolution 200nm / 0.2um
No vacuum - specimens can be alive/dead
Stained with eyes
Viewed with eye
View whole cells or tissues
Properties of scanning electron microscope:
Uses…
Magnification
Resolution
Dead / alive specimen
Stained with…
Viewed with…
Image colour…
• Uses electrons from electron gun, focused by electromagnets
• magnification x100 000 -x500 000
• resolution 3-10nm / 0.002um
• need vacuum - dead specimen
• stained with heavy metals
•Viewed on computer
• B&W
Properties of transmission electron microscope:
Uses…
Magnification
Resolution
Dead / alive specimen
Stained with…
Viewed with…
Image colour…
Uses electron from electron gun, focussed by electromagnets
•Magnification - x500 000 - x2000 000
•Resolution - 0.2 - 0.5nm / 0.0002um
• need vacuum - dead specimen
• stained with heavy metals
• Viewed on computer
• B&W
Features of light microscope images
• Light background
• 2D
• Variety of colours
• Show details larger than 200nm only
• Large organelles only - (nuclei, vacuole, cholroplasts, mitochondria) / parts of organisms/whole cells
• observing small organisms
Features of laser scanning confocal microscope
• 2D computer can make 3D
• black background
• limited colour
• observing stained structures within cells/whole cells
Features of transmission electron microscope
• Always 2D
• only grayscale unless false colour
• v high magnification
• high resolution - grainy image
• observing organelles within cells or whole cells
Features of scanning electron microscope
• 3D (inside and outside)
• outside surfaces
• greyscale/ false colour
Identify the type of microscope used when shown a photomicrograph
Light: light background
TEM: false colour, v high resolution
SEM: black background, false colour, 3D looking, v high resolution
Laser scanning confocal: black black ground, limited colour
Timeline showing development of microscope
Romans experiment with glass lenses
13th century invention of spectacles
15th century century telescope
1609 Galileo Galilei first ‘thing’ to be called microscope (compound light)
How did the development of the microscope intifuence scientific thinking.
Allowed cells to be observed - disproved spontaneous generation
Allowed theories to be presented and disproved then replaced when technology improves
Describe and explain the characteristics of a good slide preparation
No artefacts
Thin
Transparent
Clean slides + coverslips
Good contrast
Cells spread out with limited overlap
Appropriate vowme of liquid
Why do slide preparations need to be thin
To let light through
Individual cells can be identified
Why is staining useful for light microscopy
Many specimens are transparent
Some parts of the object take up more dye
Creates a contrast in colour or intensity between objects and their surroundings
Different stains bind to different molecules
What is a polychromatic stain?
Polychromatic stains show different colours with different molecules
Can more than one stain be used at once?
Yes, because they bind to different molecules
Give 4 examples of stains
Methylene blue
eosin
Toluidine blue
Iodine in potassium iodide
What do eosin, methylene blue, Toluidine blue
Iodine in potassium iodide stain ?
Methylene blue - DNA
Eosin- cell cytoplasm
• Toluidine blue - phloem - red, xylem - blue, DNA-blue
• Iodine in potassium iodide - starch blue / black
What properties does a stain need to have to be use for light microscopy?
Differential staining - stain some objects and not others
↳ contrast visible
Intense colour - v. Small amount of biological material but has to be visible
What is the formula for magnification ?
Magnification = image size / actual size
How to convert from
mm —> um —> nm
mm —> um —> nm
x1000 x1000
Define resolution
The ability to distinguish between 2 separate points
Define magnification
Number of times larger the image is compared to the object
What is the difference between magnification and resolution?
Magnification is enlargement not always increased detail
Resolution is distance
Magnification has no units
Resolution and max magnification of all the microscopes
Light : 200nm and 1500-2000x
SEM: 3-10nm and 100,000 - 500,000x
TEM: 0.2nm and 500,000 - 2,000,000
