Module 2 - LMs, Magnification, and Resolution

Question 1

How does a LM work?

Answer 1

• LM use light as a radiation source.
• The light is reflected up through the specimen.
• The light rays then travel through the objective lens, then the body tube, and then through the ocular lens.
• The image is magnified by both the objective and ocular lenses.
• The objective lens also resolves the image as well as magnifying the specimen.
• As white light passes through these coloured parts, some of the wavelengths of light are absorbed.
• Therefore, the light that reaches the other side has some wavelengths missing and appear coloured. The coloured light is then focused and detected in the eye.

Question 2

Preparing a Specimen: Sectioning

Answer 2

-Use a microtome to cut fine slices of embedded specimen so that light can pass through the specimen.

Question 3

Preparing a Specimen: Differential Staining

Answer 3

• Improves contrast between different tissues and/or structures.
• Can be permanent or temporary.

Question 4

Advantages of LMs

Answer 4

• Low skill set needed by user.
• Can be transported to use in field work.
• Can observe living organisms.
• Relatively inexpensive so available for schools and colleges.

Question 5

Disadvantages of LMs

Answer 5

• Lower resolution than EMs.
• Hence limited magnification (x1500).
• Many internal cellular structures can’t be seen. e.g. ribosomes, cristae.

Question 6

Define Magnification

Answer 6

• Number of times larger an image appears when compared to the actual specimen i.e. enlargement.
• This enables further detail to be seen.

Question 7

More Info on Magnification

Answer 7

• Achieved by the ocular lens (x10) and the objective lens (x4, x10, x40).
• Magnification is limited by resolution of the microscope.
• Total Magnification = ocular lens magnification x objective lens magnification
• Maximum magnification of LM = x1500.
• EMs can achieve higher magnifications.

Question 8

Define Resolution

Answer 8

-Ability to distinguish between two separate points. i.e. determines clarity.

Question 9

More Info on Resolution

Answer 9

• Limit of resolution = wavelength/2
• Maximum resolution of LM = 200nm (as shortest wavelength of light = 400nm).
• Hence objects closer than 200nm apart will be seen as one object.
• EMs have higher resolution as they use a beam of electrons which has a shorter wavelength. (approx that of X-rays).

Question 10

Magnification Equations:

Answer 10

Actual = Image/Magnification
Magnification = Image/Actual
mm - μm - nm
x1000 x1000

Question 11

Calculating Magnification:

Answer 11

1. ) Measure the scale bar in mm.
2. ) Convert to μm = this is the size of the IMAGE.
3. ) Find actual size using scale bar number.
4. ) Substitute known values into formula.