3.2.1.3 Microscopes

What are the 3 key types of microscopes?

• Optical (light) microscope

• Transmission electron microscope

• Scanning electron microscope

Define magnification

The magnification of a microscope refers to how many times larger the image is compared to the object.

Define resolution

The resolution of a microscope is the minimum distant between two objects in which they can still be viewed as separate.

The resolution in an optical microscope is determined by the wavelength of light

The resolution in a electron microscope is determined by the wavelength of the beam of electrons

Key details of Optical(light) microscope:

• A beams od light is condensed to create the image

• Poorer resolution (light has longer wavelength)

• Lower magnification

• Colour images

• Can view living samples

Key details of Electron microscope (SEM&TEM):

• A beam of electrons is condensed to create the image (electromagnets)

• Higher resolution (electrons have shorter wavelength)

• Higher magnification

• Black & white images

• Samples must be in a vacuum, so non-living

How does the image produced differ in TEM and SEM?

• In the transmission electron microscopes image is in 2D & shows detailed images of internal structure of cells

• In scanning electron microscopes image is in 3D

How are electrons used in the TEM?

An electron gun produces a beam of electrons that pass through the specimen.

(Parts that absorb electrons appear dark)

How are electrons used in the SEM?

Electrons are beamed onto the surface and the electrons are scattered in different ways depending on the contours.

What are limitations of the TEM & SEM?

• samples need to be extremely thin (TEM)

• image may contain artefacts

• result is flat and 2D (TEM)

• complex staining process is required

• SEM has lower resolving power than TEM

What is the magnification formula?

I=AM

How can the actual size of structures be measured?

Using the eyepiece graticule scale.

What is cell fractionation?

A method used to isolate different organelles so they can be studied.

How does Cell fractionation happen?

Cells are broken open to release the contents and organelles are then separated

What are the three properties the solution needs to be in cell fractionation (and why)?

• Cold - to reduce enzyme activity so organelles don’t get damaged

• Isotonic - to keep same water potential to prevent osmosis which could cause organelles to burst/shrivel

• Buffered - so pH doesn’t fluctuate which prevents damage to organelles

Whats the two steps to cell fractionation?

Step 1. Homogenisation

Step 2. Ultracentrifugation

What is the method of cell fractionation?

• Cell must be broken open (homogenised) using a blender (in the solution)

• Solution is filtered to remove large cell debris

• The filtered solution is spun at different speeds in a centrifuge

• Organelles separate according to their densities

What is the order the organelles separate out?

• Nuclei (Most dense)

• Chloroplast (if using plant tissue)

• Mitochondria

• Lysosomes

• Endoplasmic reticulum

• Ribosomes (Least dense)