Microscopy Flashcards
(20 cards)
What is magnification?
How many times larger an image is than a object
What is resolution?
Ability to distinguish between 2 separate points
How do you calibrate an eye piece graticule?
1) fix stage micrometer into place on stage
2) looking through eyepiece line up graticule and stage micrometer
3) count number of graticule divisions that fit into 1 micrometer division
4) use formulae to calculate length of graticule division
What is the formulae to calculate the length of a graticule division?
size of micrometer division ÷ number of graticule divisions
What is the resolution and magnification of a light microscope?
Resolution = 0.2µm
Magnification = x1,500
How do you produce a wet mount slide?
1) pipette a small drop of water on centre of glass slide
2) Using forceps place thin section of the specimen onto the drop of water
3) Add few drops of stain (e.g iodine) to the specimen
4) Slowly add a cover slip onto specimen
What is the purpose of a dye in light microscopy?
To increases contrast and allows cell components to become visible
What are the 3 other methods of preparing slides and how do they work?
Dry mount = specimen placed onto the slide and covered with cover slip
Squash slide = wet mounts prepared and cover slip is pressed to squash the cells
Smear slide = edge of slide used to smear sample to create a thin, even coating on a separate slide
How do you view a microscope slide using a light microscope?
1) clip prepared microscope slide onto stage
2) select the objective lens with lowest power
3) use coarse focus to bring stage just below objective lens
4) look down eyepiece and use coarse focus to move stage downwards until the image is roughly in focus
5) use fine focus to make the image clearer
6) If higher magnification is needed swap to more powerful objective lens and refocus
What is an electron microscope and what type of image does it create?
Uses electrons to form an image of a dead/non-living sample in black and white
What is a transmission electron microscope?
Uses electromagnets to transmit electron beam through specimen (denser parts absorb more electrons so appear darker in image formed)
What type of image does a TEM produce?
2D black and white image
What is the resolution and magnification of a TEM?
Resolution = 0.5 nm
Magnification = x 1,500,000
What is a scanning electron microscope?
Scans a beam of electrons across specimen surface and reflected electrons form an image
What type of image does a SEM produce?
3D black and white image
What is the resolution and magnification of a SEM?
Resolution = 5 nm
Magnification = x 1,500,000
What is a laser scanning confocal microscope?
Type of fluorescence microscopy using laser beam (single focused light spot) to scan a specimen that’s been tagged with a fluorescent dye
How does a laser scanning confocal microscope create an image?
Dyed components give off light which is focused through pinhole onto a detector connected to a computer which generates an image
this image can then be converted into a 3D image
What is the resolution and magnification of laser scanning confocal microscopes?
produce clearer images than a light microscope however have a lower resolution than electron microscopes
What can laser scanning confocal microscopes be used for?
- look at different depths within specimen
- observe living samples
