2.1.1 microscopy Flashcards
(32 cards)
how does a light microscope work?
they direct light through a sample on a glass slide
the image is focused using lenses + is visible through the eyepiece
explain why the LM has two types of lenses?
the objective lenses will magnify the sample image and the eyepiece lenses magnifies it again
this allows for a higher overall magnification
what is the magnification and resolution of the LM?
resolution is about 0.3 micrometers
magnification is about x1500
this has the worst mag and resolution
what are the advantages of a LM?
you can view dead and living specimens
it’s good for viewing whole cells or organisms
you can see natural color
they are small and portable
easy use and simple preparation
cheapest equipment
what are the disadvantages of a LM?
low magnification and resolution (wavelength of light)
cannot see internal structure
how do you calibrate an eyepiece graticule?
- line up graticule and stage micrometer while looking through eyepiece
- count the no. of E.G divisions in 1 division of S.M
- calc the ration {remember 1 S.M division = 1 micrometer}
- calc how much one division on E.G is worth
how do transmission electron microscopes (TEM) work?
electron beams are fired w/ an electron gun through the specimen
some electrons get absorbed; the more that get absorbed the darker it appears
electromagnets used to focus
views through fluorescent screen
done in a vacuum
why must TEM specimens be in a vacuum?
to prevent diffraction of electron when they hit air particles
must be viewed on a copper grid (glass = diffraction)
what is the mag + resolution of TEM?
they have the BEST mag and res.
you can see internal structure
due to electrons having a shorter wavelength so specimens can be closer before they diffract
what are the advantage of TEM?
you can see internal structure
very high resolution and magnification
can be artificially colored
what are the disadvantages of TEM?
the image is in 2D and black and white
cannot view live specimens (vacuum)
expensive equipment
need special skill to operate
not portable and heavy
extensive sample prep process
how do scanning electron microscopes (SEM) work?
a beam of electrons is fired across the surface of the sample
the electrons scatter off
the different depths in the sample create 3D images
what are the advantages of SEM?
can see 3D images
can view thicker specimens
what are the disadvantages of SEM?
can only view dead specimens (vacuum)
heavy and not portable
expensive equipment
complex sample prep
samples are artificially colored
what is the mag and resolution of the SEM?
they have the 2nd best mag and resolution
not as high as TEM
how do you prepare slides for all 3 microscopes?
LM = can have wet and dry mount; squash and smear slides; use glass slide and coverslip; can be stained; extremely thin specimens needed
TEM = stained with heavy metals; set in resin and stained again
SEM = fractured and stained with heavy metals
what are artifacts and how are they created?
visible structural detail caused by processing of a specimen and NOT a part of the specimen itself
can occur in light and electron microscopes
ie bubbles, loss of membrane continuity, empty space in cytoplasm
how do laser scanning confocal microscopes (LSCM) work?
flood the specimen with a laser (powerful beam of light)
mirrors are used to focus it
the laser will illuminate fluorescent stains on spec allowing them to be seen
what is the mag and resolution of a LSCM?
they have a high mag and res. due to the pinhole which blocks out of focus light
res. around 800nm
in between LM and EM
what are the advantages of LSCM?
they have a high mag and res (pinhole)
can view thicker specimens
able to create 2D and 3D images (stacking)
can see dead and living specimens
what are the disadvantages of LSCM?
laser can damage cells in living specimens
more expensive than LM
complex’s set up and sample prep
not portable
creating stacks is time consuming
special skill required
how is staining used in light microscopes?
normally specimens are transparent so dyes absorb light and allow more visibility
dyes absorb some colors + reflect others
certain tissues absorb certain dyes
which stains are commonly used in LM?
toluidine blue (blue cells)
phloroglucinol (pink/red cells)
how is staining used in EM?
electrons have no color so stains are needed
heavy metal compounds absorb electrons well
makes specimen black and diff shades of grey
