1- Microscopes

Describe a Light microscope

• 2 lenses (Objective and eyepiece) to produce image

• Total magnification = objective x eyepiece

  • Magnification- number of times larger an image appears

• Resolution- ability to distinguish between 2 points on the same specimen as separate

• 1 billion nanometers (nm)= 1 metre (m)

What are the advantages on a light microscope?

• Cheaper

• Easier to move

• Takes up less space

• Easier to use

• Live specimens

What are the disadvantages of a light microscope?

• Resolution limited by wavelength of light

• 2 objects may be seen as one

• Artefacts may be present- left from staining process or byproduct/ structure that shouldn’t be there

Why do we stain?

• Add contrast

• Show against light background

• Helps identify different cell structures

Describe an electron microscope

• Uses beam of electrons and focused with electromagnets

• Small wavelength- less than 1 nm

• Use in a vacuum so trajectory of electrons doesn’t change

What are the advantages of an electron microscope?

• Higher max magnification

• Smaller objects can be seen

• Can see cell ultra structure

• Upon to 2 million magnification

What are the disadvantages of an electron microscope?

• Large and expensive equipment

• Specimens must be dead

• Stains are hazardous

• Skill and training is necessary to prepare slides

• Artefacts may be present

Electron microscope: Transmission

• Dehydrated with solvent and chemically fixed

• Set in resin and cut with microtome

• Electron beam fired through vacuum at specimen which is stained with heavy metals and salts

• Electrons which pass through are focused onto a screen

• 2d black and white image produced

• Up to 2 million magnification

• 0.5 nm resolution

Electron microscope: scanning

• Electrons bounce off specimen

• Must be in vacuum and coated in metal

• Gives 3D image of surface contours

• Black and white but false couloir can be added

• Up to 2 million magnification

• 3-10 nm resolution

Setting up EMs

Slides:

• Dehydrated

• Dead specimens

• Chemically fixed

• Stained with metals

The microscope:

• Beam of electrons

• Focused with electromagnets

• Used in vacuum so trajectory of electrons doesn’t change

What are the 4 types of mounting?

• Dry mount

• Wet mount

• Squash slide

• Smear slide

Dry mount

• Solid specimens viewed whole or cut into thin sections

• Specimen placed in middle of slide and cover slip placed on top

• E,g, hair, pollen and dust

Wet mount

• Specimen suspended in an aqueous solution

• Cover slip placed at angle to avoid air bubbles

• Aquatic samples can be viewed this way, it views living organisms

Squash slide

• Wet mount prepared and lens tissue used to gently press cover slip placed

• Care needs to be taken not to break the cover slip

• Root tip squashes are sued to observe mitosis

• Good for soft samples

Smear slide

• Drop of sample placed on

• Edge of another slide used to smear sample, creating a thin, even coating

• Cover slip placed over sample

• Often used to view blood samples

Staining

• Stains are coloured chemicals which bind to molecule sin or on a specimen

• Biological specimens contain lots of water so have little contrast as they don’t absorb much light

• Some stains bind to specific cell structures

• Some are general (e.g. methylene blue)

• Use of different stains for different structures is differential staining

• Staining increases contrast, making specimens easy to see

Preparing a sample to stain

• Place specimen on slide and allow to air dry

• Heat fix by passing through a flame

• Specimen will adhere to the slide and take up stains

Examples of stains and their uses:

• Acetic orcein- binds to DNA and stains chromosomes dark red

• Eosin- stains cytoplasm

• Sudan red- stains lipids

• Iodine- stains cell walls in plants yellow and starch blue black

Preparing a slide

1) Fixing- chemicals used to preserve specimens

2) Sectioning- Dehydrated with alcohol, placed in wax or resin, thinly sliced with a knife

3) Staining- Differential staining used to show different structures

4) Mounting- Specimen secured to microscope slide and cover slip placed on top

Rules for drawing from a specimen on a slide

• Descriptive title with magnification total

• Represents what you see (proportional)

• Solid continuous lines (no sketching)

• Closed cells

• No shading

• Label lines with ruler

• Label lines touch the feature it is describing

• No arrow heads

• No overlapping