Microscopy

Parts of the microscope Ocular lens Revolving nosepiece or turret Arm Objective lens Stage Iris wheel diaphragm Coarse focus Light source Fine focus

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 minigrid Each 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 As magnification increases, field of view decreases by the same factor x40 x100 x400 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 ruler Measure scale with ruler Use scale to convert ruler measurement of object to real one Size = 0.6mm = 600 µm

Size of objects under the microscope 1 example 1.7 mm ~1.7 mm ~1200 m

Size of objects under the microscope 2 If given a field of view – Magnification x100, field of view 2000 m Measure object with ruler Measure field of view with ruler Use 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 view So size of each cell = 1000 m

Size of objects under the microscope 3 If given a magnification – Magnification x 200 Measure object with ruler Divide 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 m Cell is about ½ of field of view = 750m

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 =

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

How big are these? 1. Magnification x100, field of view 1600 m ~2 fit across field so ~800 m 2. Magnification x400, field of view 200 m ~5 fit across field so ~40 m