Microscopy Flashcards
How do you use a light microscope?
- take a thin slice of specimen, place in the middle of the slide
- place a coverslip on top at a 45 degree angle, to prevent air bubbles obstructing view
- arms used to clip the slide in place
- rotate turret with objective lens to the lowest (x4 mag)
- looking through eye piece, use fine adjustment knob until a clearer image can be viewed
- rotate the objective lenses and select x10 power, followed by the fine knob
How to calculate magnification?
Image size / actual size
What is the definition of magnification?
no. of times greater that an image is than the object
What is the definition of resolution?
the ability to distinguish between two objects that are very close together, so better detail can be seen
Why do we stain things?
It provides contrast because different parts of the cell take up different volumes of the stain
How to prepare a dry mount?
- cut samples into thin slices, using sharp blade = known as sectioning
- cover with a cover slip so it is ready to be viewed e.g hair, pollen, insects
How to prepare a wet mount?
specimen is covered with a drop of liquid such as water, oil or stain
What are squash slides?
where the sample is squashed between the slide and the coverslip (a type of wet mount) = stain that is used for soft tissue such as root tips
What are smear slides?
where another slide is used to smear the sample across the slide (the liquid spreads out), used for a thin layer of liquid e.g blood
What are the advantages and disadvantages of using a light microscope?
+ easy to use
+ color image
+ cheap, accessible
- low magnification and resolution
- can’t see organelles e.g. ribosomes
What is a scanning electron microscope? How does it work?
Where electrons spread out on the surface of the specimen, then reflected back off the surface
(wavelength of the electrons is shorter than light)
What are the advantages and disadvantages of using a scanning electron microscope?
+ forms 3D shape
+don’t need it to be a thin sample
- done in a vacuum = no live samples can be used
- cannot see the internal structure
- lower magnification and resolution that transmission electron microscope (T.E.M)
How does a transmission electron microscope work?
Uses a beam of electrons that pass through the specimen
What are the advantages and disadvantages of using a transmission electron microscope?
+ better resolution
+ shorter wavelength of electrons than light
+allows internal structures to be seen
- can’t look at living organisms
- doesn’t form a colour image
- 2D
- needs to be ultra thin specimen to allow electrons to pass through
How does a laser scanning confocal microscope work?
-> fluorescent dye is added to the specimen
-> laser beam (light) focused on part of the cell
->light fluorescents from different depths within the cell
-> a pinhole sits infront of the detector
->pinhole eliminates out of focus light
