microscopy Flashcards
describe what a microscope does.
a microscope is an instrument that allows you to magnify objects, making individual cells visible etc.
name 4 different types of microscope.
- light microscope
- scanning electron microscope
- transmission electron microscope
- laser scanning confocal microscope
what do the abbreviations TEM and SEM stand for?
TEM - transmission electron microscope
SEM - scanning electron microscope
outline how an SEM works.
they fire a beam of electrons through a vacuum onto a sample. the electrons are bounced off the sample and detected to produce a 3D image.
outline how a TEM works.
fire a beam of electrons through a vacuum onto a sample. the electrons pass through the very thin sample. it passes through dense areas less easily. the electrons are detected underneath the sample to produce a 2D image.
outline how a laser scanning confocal microscope works.
a single spot of light is passed across a sample, the dye within the sample causes fluorescence. this is filtered back through a pinhole aperture to the eyepiece.
outline how a light microscope works.
it has two lenses, the objective lens near the sample and the eyepiece lens. the two lenses magnify the image. the sample is lit from below .
compare the uses of the different microscopes.
light - living or dead sample, staining is used, cheap and most common.
TEM - dead sample. preparation can cause damage. expensive and hard to use. 2D image produced
SEM - similar to TEM but produces 3D images
SLCM - used to view living and moving samples. difficult to stain, provides good contrast.
compare the magnification and resolution of the types of microscope.
light - magnification = x2000. resolution = 200nm
SEM - magnification = x500000. resolution = 3-10nm
TEM - magnification = x500000. resolution = 0.5nm
define the term resolution
the ability to distinguish two points close together as being separate and to see detail.
define the term magnification.
the extent to which an object has been enlarged.
state the magnification formula.
magnification = size of image/actual size of object.
what is the formula for the overall magnification of an image?
overall magnification = objective lens x eyepiece lens.
explain how to convert units when calculating magnification.
1000 nm = 1 um
1000 um = 1 mm
when measurements are getting smaller divide by 1000. when getting bigger multiply by 1000.
why is staining useful in light microscopy?
cell structures are often transparent. stains increase contrast as different components of the cell will stain to a different degree. components become more visible and easier to identify.
explain how samples are stained in light microscopy.
- first the sample is placed on a slide and allowed to dry.
- it is heat - fixed by passing it through a flame.
- the specimen will stick to the microscope slide and then take up the stain when it is added.
name two different stains.
methylene blue stains parts of the cell whereas Congo red stains the background making the cell stand out.
describe how to produce a temporary wet mount.
place the cover slip on a 45 degree angle on the edge of the droplet. drop the slip gently. trying to avoid air bubbles.
why do slide preparations need to be thin?
so that the layers of cells in the specimen are minimised so individual cells are clearer.
what is an eyepiece graticule?
a glass disk marked with a 1 - 100 scale. it has no units and remains unchanged no matter the magnification. the relative sizes of the divisions increases with each increase in magnification.
what is a stage micrometer?
a microscope slide with a very accurate scale in micrometres engraves on it. the scale is 100 divisions = 1 mm so 1 division = 10 micrometres.
explain how to calibrate a 4 x objective lens..
- put the stage micrometer and eyepiece graticule in place and focus the scale on the micrometer slide.
- align the micrometer scale with the scale in the eyepiece.
- x divisions on the eyepiece graticule = n divisions on the stage micrometer.
- 1 graticule division = number of micrometres/number of graticule divisions.
= magnification factor.
