Microscopy

Parts of the microscope

Ocular lens

Arm

Coarse focus

Fine focus

Objective lens

Stage

Iris wheel diaphragm

Light source

Revolving nosepiece or turret

Using the microscope

• Change the magnification by

Changing the objective (using the turret) and ocular lenses

• Change the focus by

Adjusting coarse and fine focus• Adjust the light or contrast by

Adjusting iris wheel diaphragm (or condenser or mirror if present)

Light microscopy

• Can be used on living cells

• Maximum magnification ~ x 2000

• Staining can be used to make structures more visible

• What can be seen? – cells and organelles

Electron microscopy

• Can not be used on living cells• Maximum magnification x millions• Staining is often used eg silver, gold• What can be seen? – details of organelles,

large molecules

Staining

Stains are used to make some structures more visible

They work by being absorbed by some molecules more than others

Examples include iodine, methylene blue, eosin

Working distance

This is the distance between the objective lens and your slide.

The higher the magnification of the lens, the larger the lens

The higher the magnification of the lens, the smaller the working distance

What you see under the microscope

Everything is reversed as well as magnified

What you see under the microscope 2

• If the object appears to be at the top of the slide it is really at the bottom

You need to move the slide away from you

• If the object appears to be at the left of the slide it is really at the right

You need to move the slide to the right

Diameter of field of view

• Distance across centre of field

• Measured with a minigrid

• Measured in micrometres (m)

Using a minigridEach square is 1 mm by 1 mm in size

The centre grid is further subdivided into 0.1 mm grid squares

Millimetres and micrometres

1 mm = 1000 m• 1.5 mm = m

• 0.4 mm = m

• 2400 m = mm

• 350 m = mm

Millimetres and micrometres

1 mm = 1000 m• 1.5 mm = 1500 m

• 0.4 mm = 400 m

• 2400 m = 2.4 mm

• 350 m = 0.35 mm

Magnification and field of view

x40 x100 x400

As magnification increases, field of view decreases by the same factor

As magnification decreases, field of view increases by the same factor

Calculating field of view when magnification changes 1

magnification x 40 magnification x 400

field of view 4000 m field of view 400 m

Magnification has increased by a factor of 10

Field of view has decreased by a factor of 10

Calculating field of view when magnification changes 2

magnification x 100 magnification x 400

field of view 1600 m field of view 400 m

Magnification has decreased by a factor of 4

Field of view has increased by a factor of 4

Size of objects under the microscope 1

If given a scale – 0 1 2 mm

Measure object with rulerMeasure scale with rulerUse scale to convert ruler measurement

of object to real one

Size = 0.6mm = 600 µm

Size of objects under the microscope 1 example

~17 m

1.7 mm

~1200 m~1.7 mm

Size of objects under the microscope 2

If given a field of view –

Magnification x100, field of view 2000 m

Measure object with rulerMeasure field of view with rulerUse field of view measurement to

convert ruler measurement of object to real one

Size = 1000 m

Size of objects under the microscope 2 example

Magnification x40, field of view 4000 m

Cells take up roughly ¼ of field of viewSo size of each cell = 1000 m

Size of objects under the microscope 3

If given a magnification –

Magnification x 200

Measure object with rulerDivide size by magnification factor to

get real size

Size = 4cm ÷ 200 = 0.02 cm = 0.2 mm = 200 m

Size of objects under the microscope 3 example

Magnification x100, field of view for x400 = 350 m

Field of view for x100 = 1400 mCell is about ½ of field of view = 750m

Magnification and field of view

If field of view at x100 is 2000 m, then

• Field of view at x400 =

• Field of view at x40 =

If field of view at x400 = 250 m, then

• Field of view at x100 =

• Field of view at x40 =

Magnification and field of view

If field of view at x100 is 2000 m, then

• Field of view at x400 = 2000 ÷ 4 = 500 m

• Field of view at x40 = 500 x 10 = 5000 m

If field of view at x400 = 250 m, then

• Field of view at x100 = 250 x 4 = 1000 m

• Field of view at x40 = 250 x 10 = 2500 m

How big are these?

1. Magnification x100, field of view 1600 m

2. Magnification x400, field of view 200 m

How big are these?

1. Magnification x100, field of view 1600 m

2. Magnification x400, field of view 200 m

~2 fit across field so ~800 m

~5 fit across field so ~40 m