Biology: Chapter 1: Microscopy

Biology: Chapter 1: Microscopy

Units of measurement

metre	m	= 1 m
millimetre	mm	= 10-3 m
micrometre	mm	= 10-6 m
nanometre	nm	= 10-9 m

Magnification and Resolution

Magnification - How much bigger an image seems under a microscope than it does in real life

Magnification = Image size / Actual size

Resolution - The ability to distinguish two separate points in an image / How clear the image is.
 - The resolution of an image is based on the length of the wavelength used to view it. If the specimen being observed is smaller than half the wavelength used to observe it, it will not be detected as the light waves will not hit the specimen. 
- Using a microscope with a stronger magnification will not increase the resolution, it will only increase the size of the image. If the specimen is smaller than 200nm (half a light wavelength), it will only be seen as one point.
- The resolution of an electron microscope is much higher than that of a light microscope.

Light Microscopy

- Specimens are illuminated with light, which is focused with glass lenses and viewed with the eye. 

- Specimens can be living or dead, but usually need to be stained with a dye (eg. iodine) to make them more visible.

- Many different stains are available that stain different parts of the cell eg. Lipids, DNA, cytoskeletons, mitochondrion, etc. 

- Many light microscopes are compound microscopes (use many different lenses to gain a different magnification). 

- Has a resolution of around 200nm (half a light wavelength), which is enough to view cells but not the details of cell organelles. 

-Cheap and easy to operate and maintain.

-Vacuum not required.

-Natural colour maintained.

-Can only magnify images up to a maximum of 2000 times.

-Small and portable.

Electron Microscopy

-Uses a beam of electrons instead of the electromagnetic spectrum.

-Easily produced (using hot wire), focused (using electromagnets), detected (using phosphor or photographic film)

-Wavelength of less than 1nm

-Only works in a vacuum so electrons don't deflect off air molecules and other substances, so specimens can only be dead.

-Must be stained with electron-dense chemicals (eg. metals such as lead, gold)

-Artefact: Preserved structures created when processing the specimen for viewing and therefore not part of actual specimen.

-All images in black and white

-Expensive to buy and handle 

-Complicated preparation

-Requires a lot of space

-Magnifies over 500 000 times

Transmission electron microscope (TEM)

- Passes a beam of electrons through the specimen which then get detected on a fluorescent screen and displays the image, similar to light microscopes.

-Thin sections are needed for TEM because the electrons need to pass through

-Most common form of electron microscopy, has the best resolution

Scanning electron microscope (SEM)

-Passes a beam of electrons over the surface of the specimen

-Electrons reflect off the specimen which has been coated in metals, which are then focused onto a fluorescent screen to produce an image.

-Has lower resolution than TEMs, but gives 3 dimensional images of the specimen.

-Can use thicker sections of the specimen since electrons don't have to pass through.

Preparation of slide samples for light microscopes

1. Fixation: Chemicals preserve the specimen in a life like condition. Does not distort specimen.
2. Dehydration: Water removed from specimen (usually using ethanol). Especially useful for electron microscopy because the water molecules deflect the electrons, blurring the image.
3. Embedding: Supports the tissue in wax or resin so it can be cut into thinner sections, letting light or electrons go through and make the specimen easier to view.
4. Mounting: Mounting the specimen on a slide and covering it so it's protected and suitable for viewing for a long period.