2.1.1 Microscopes Flashcards
resolution definition
the degree to which it is possible to distinguish two objects, which are very close together
magnification definition
how many times bigger the image of a specimen observed is, compared to the actual size of the specimen
magnification equation
magnification=size of image/size of actual
method for using light microscope: 9 steps
- Prepare sample using a glass slide, coverslip and stain
- Plug microscope into plug socket and turn on
- Turn light on
- Put sample on the stage, secure with clips
- Adjust to lowest magnification
- Move stage all the way up
- Use the coarse focus wheel to get sample into focus (larger field of view)
- Move to next objective lens (use fine focus wheel to show detail)
- Repeat with next objective lens
light microscope: how does it work?
it works by passing light through a specimen and focusing the light on an eyepiece or camera, which magnifies the image.
light microscope: what is it used for?
-schools and colleges
-hospitals
-universities
light microscope: preparation of specimen
place a slide with cover slip and with a stain
light microscope: maximum magnification
x1500
light microscope: 2D or 3D, colour or black and white
2D, colour
light microscope: resolution
0.2 μm
light microscope: advantages 4
-relatively cheap
-easy to use
-portable
-able to study whole living specimen
light microscope: disadvantage 1
-resolution is limited (as cannot increase magnification any higher with detail)
confocal laser scanning microscope: how does it work?
a thick section of tissue or small living organisms are scanned with a laser beam
the laser beam is reflected by the fluorescent dyes
multiple depths of the tissue section/organisms are scanned to produce an image
(think of it like the laser beam is building up the image layer by layer)
confocal laser scanning microscope: what is it used for?
-medical professions
-branches of biological research
confocal laser scanning microscope: preparation of specimen
the cells being viewed must be stained with fluorescent dyes
confocal laser scanning microscope: max magnification
not needed
confocal laser scanning microscope: 2D or 3D, colour or black and white
3D, colour
confocal laser scanning microscope: resolution
do not need
confocal laser scanning microscope: advantages 4
-they can be used on thick or 3-D specimens
-they allow the external, 3-D structure of specimens to be observed
-very clear images are produced, the high resolution is due to the fact that the laser beam can be focused at a very specific depth
confocal laser scanning microscope: disadvantages 2
-it is a slow process and takes a long time to obtain an image
-the laser has the potential to cause photodamage to the cells
transmission electron microscope (TEM): how does it work?
use electromagnets to focus a beam of electrons, this beam of electrons is transmitted through the specimen
denser parts of the specimen absorb more electrons tf denser parts appear darker on the final image produced (produces contrast between different parts of the object being observed)
transmission electron microscope (TEM): what is it used for?
-biological research
transmission electron microscope: preparation of specimen
the specimen have to be chemically fixed by being dehydrated and stained with metal salts, stained in order to absorb the electrons (heavy metal compounds are often used)
transmission electron microscope (TEM): max magnification
up to x 2 million
transmission electron microscope (TEM): 2D or 3D, colour or black and white
2D, black and white
transmission electron microscope (TEM): advantages 2
-high resolution
-this allows the internal structures within cells (or even within organelles) to be seen
transmission electron microscope (TEM): disadvantages 6
-specimen cannot be alive (as there is a vacuum inside of TEM, all water must be removed from specimen)
-large
-very expensive
-need a great deal of skills and training to use
-they can only be used with very thin specimens or thin sections of the object being observed
-the lengthy treatment required to prepare specimens means that artefacts can be introduced (artefacts look like real structures but are actually the results of preserving and staining)
scanning electron microscope (SEM): how does it work?
SEMs scan a beam of electrons across the specimen
This beam bounces off the surface of the specimen and the electrons are detected, forming an image
Tf SEMs can produce three-dimensional images that show the surface of specimens
scanning election microscope (SEM): what are they used for?
biological research
scanning electron microscope (SEM): preparation of specimen
specimen has to be placed in a vacuum and often coated with a fine film of metal
scanning electron microscope (SEM): max magnification
up to 200,000x
scanning electron microscope (SEM): 2D or 3D, colour or black and white
3D, black and white
(the computer programme can add false colour
what type of instruments are microscopes?
optical
what scale is used for microscopes?
logarithmic
what does logarithmic mean?
it goes up in steps, where each is 10-fold increase
what is the effect of increasing magnification,on resolution?
increasing magnification, doesn’t increase resolution
why does light limit resolution in light microscopes?
it is impossible to resolve two objects that are closer than half the wavelength of light
(the wavelength of visible light is between 0.5 or 0.65),
what can a light microscope observe?
eukaryotic cells, their nuclei and possibly mitochondria and chloroplasts
scanning electron microscopes (SEMs): 2 advatages
-they can be used on thick or 3-D specimens
-they allow the external, 3-D structure of specimens to be observed
scanning electron microscopes (SEMs): 3 disadvantages
-they give lower resolution images than TEMs
-they cannot be used to observe live specimens
-they do not produce a colour image
what should be used to calibrate a microscope?
a stage micrometer
