2.1Microscopy (CP1-practical)

Question 1

Why do samples need to be stained for microscopy?

Answer 1

Stain binds to structures
Heavy metals- electron microscopes
Coloured dye- optical microscopes
Facilitates absorption of electrons/ wavelengths of light to produce image

Question 2

Define magnification and resolution

Answer 2

Magnification- factor by which the image is larger than the actual specimen

Resolution- smallest separation distance at which 2 separate structures can be distinguished from one another

Question 3

State the magnification and resolution of a compound optical microscope

Answer 3

Magnification x2000
Resolution 200nm

Question 4

How does an optical microscope work?

Answer 4

1. Lenses focus rays of light and magnify the view of a thin slice of specimen
2. Different structures absorb different amounts and wavelengths of light
3. Reflected light is transmitted to the observer via objective lens and eyepiece

Question 5

What limits the resolution of an optical microscope?

Answer 5

Diffraction of light

Any structures closer than half the wavelength of light cannot be distinguished as separate

Question 6

Describe how transmission electron microscope (TEM) works.

Answer 6

1. Pass a high energy beam of electrons through thin slice of specimen.
2. More dense structures appear darker since they absorb more electrons.
3. Focus image onto fluorescent screen or photographic plate using magnetic lenses.

Question 7

State the magnification and resolution of a TEM

Answer 7

magnification: x500 000

Resolution: 0.5nm

Question 8

Describe how a scanning electron microscope (SEM) works

Answer 8

1. Focus a beam of electrons onto a specimen’s surface using electromagnetic lenses
2. Reflected electrons hit a collecting device and are amplified to produce an image on a photographic plate.

Question 9

State the magnification and resolution of SEM

Answer 9

Magnification x500 000
Resolution 3-10 nm

Question 10

Explain how to use an eyepiece graticule and stage micrometer to measure the size of a structure.

Answer 10

1. Place micrometer on stage to calibrate eyepiece graticule.
2. Line up scales on graticule and micrometer. Count how many graticule divisions are in 100um on the micrometer.
3. length of 1 eyepiece division = 100um / number of
   divisions
4. Use calibrated values to calculate actual length of structures.

Question 11

State an equation to calculate the actual size of a structure from microscopy.

Answer 11

I= A/M

Image size= actual size/ Magnification