microscopes and microscopy

why is a stain used when preparing a slide?

to enhance the visualisation of cells by adding contrast

what is resolution?

the minimum distance apart that two objects can be for them to appear as separate items

the greater the resolution the greater the…

clarity of image produced

what is magnification?

how many times bigger the image is compared to the object

what is the equation for magnification?

magnification = image size / actual size

how do you calculate magnification using a scale bar?

1. measure scale bar image in mm

2. convert to micrometers by x by 1000

3. use equation magnification = scale bar length in image (in micrometers) / actual scale bar length (written on scale bar)

light/optical microscopes

• use a pair of convex glass lenses to project magnified image of an object onto the eye

• use stains to enhance visualisation of cell components by adding contrast

what is the max resolution of a light microscope?

0.2 micrometers

why is the resolution of a light microscope low?

light microscopes are limited by the wavelength of light

what is the max magnification of a light microscope?

1500x

electron microscopes

• use beams of electrons that are focused by electromagnets inside a vacuum environment

• stained with a metal

why is a vacuum environment required while using an electron microscope?

so particles in the air don’t defect the electrons out of the beam alignment

what is the max resolution of an electron microscope?

0.1 micrometers

what is the max magnification of a electron microscope?

2,000,000x (2×10⁶)

why do electron microscopes have a higher resolution then light microscopes?

because electrons have a shorter wavelength than light

a higher resolution means…

a higher magnification can be used, allowing us to view smaller organelles

what are the two types of electron microscopes?

• scanning electron microscopes (SEM)

• transmission electron microscopes (TEM)

how does a SEM work?

• a beam of electrons is passed across the surface of the cell and scatter

• the pattern of scattering builds up a 3D image

• specimens are coated with a thin layer of metal

how does a TEM work?

• a beam of electrons passes through a thin section of a specimen

• denser areas absorb more electrons and appear darker on the electron micrograph produced

• specimens are stained using heavy metals

why is the contrast created?

electrons can’t pass through the heavily stained areas as they’re absorbed

what are the advantages of a TEM?

• has a high resolving power (than optical and SEM) so therefore higher magnification

• can view organelle internal structures

what are the disadvantages of a TEM?

• must be in a vacuum environment

• staining process is complex

• heavy staining can produce artifacts

• specimen must be very thin and dead

what are artifacts?

visible detail that is not part of the specimen

what are the advantages of SEM?

• specimen doesn’t have to be thin or dead

• 3D images are created

what are the disadvantages of a SEM?

• must be in a vacuum environment

• staining process is complex

• have a lower resolving power than TEM

why does the specimen need to be thin when preparing a slide?

to allow light or electrons to pass through