Calibration & Types of Microscopes

Question 1

Outline how you would prepare a temporary mount of tissue for an optical microscope [4 marks]

E.G.
How would you prepare an onion cell slide?

Answer 1

1) Pipette a drop of WATER onto a slide.

2) Use tweezers/FORCEPS to separate epidermis tissue from onion, and place the tissue onto the slide.

3) Pipette a drop of a STAIN (E.G. iodine dissolved in potassium iodide)

4) Lower cover slip gently on top using a MOUNTED NEEDLE.

5) Place slip onto stage of microscope and clip it.

Question 2

Explain what is meant by ‘resolution’ [1 mark]

Answer 2

Smallest distance between 2 points of an image that can be clearly distinguished.

Question 3

Explain what is meant by ‘magnification’ [1 mark]

Answer 3

The extent to which an image is enlarged and seen through the lens clearly.

Question 4

Explain what is meant by the term, ‘calibrate’ [1 mark]

Answer 4

Arranging an instrument to provide a result for a sample within an acceptable range.

basically…
the size of the eyepiece graticule units using a stage micrometer.

Question 5

What is the formula for magnification?

Answer 5

Magnification = eyepiece x objective lens

Question 6

Explain why it was important that the sections of onion tissue were thin [2 marks]

Answer 6

A thin section allows more LIGHT to be passed through (1)

…which allows a SINGLE LAYER of cells to be viewed (1)

Question 7

A student investigated mitosis in the tissue from an onion root tip. She prepared the slide but was then given an extra instruction.

“Push down hard on the cover slip, but do not push the cover slip sideways”

Explain why she was given this instruction. [2 marks]

Answer 7

Push hard to squash the tissue to create a single layer of cells. (1)

Do not push sideways as this will cause them to roll together and crease. (1)

Question 8

What is an artefact? [1 mark]

Answer 8

Something observed in a microscopic image that is not naturally present (and not part of the cell) but occurs as a result of the procedure.

Question 9

Explain how you would calibrate the size of an eye piece graticule, and find out the length of a specimen [4 marks]

Answer 9

1) Place stage micrometer on the microscope stage.

2) Line up the divisions on eyepiece graticule scale with micrometer scale.

3) Work out length of 1 eyepiece graticule units in micrometers.

4) Measure sample using eyepiece graticule at SAME magnification.

5) Calculate the actual length of the cell.

Question 10

How would you work out the length of one eyepiece graticule unit if…

100 units on eyepiece graticule = 3mm on stage micrometer

Answer 10

100 units on eyepiece graticule = 3mm on stage micrometer

100 units = 3000 micrometers.

1 unit = 30 micrometers.

Question 11

What is the formula to work out the actual size of a cell?

Answer 11

Magnification = Image size / Actual size

      I    M       A                    = MIA = Missing In Action

     I    A        M                  = IAM = I am

Actual size = Image size / magnification

Question 12

What are the 2 types of microscopes?

Answer 12

> Optical microscope

> Electron microscope

Question 13

What are the 2 types of electron microscopes?

Answer 13

> Transmission electron microscope (TEM)

> Scanning electron microscope (SEM)

Question 14

What are the differences of pros and cons of TEM and SEMS? [4 marks]

Answer 14

For TEMS…
+ Highest resolution
- Specimen must be extremely thin - T for thin
- 2D image

But for SEMS…
+ Specimen does not need to be thin.
+ 3D image
- Only shows outer surface.

Question 15

What are the strengths and limitations of using an optical microscope?

Answer 15

+ Colour image
+ Can use living specimens
+ Cheaper - affordable apparatus.

• 2D image
• Lower magnification / resolution compared to electron microscopes.

Question 16

Describe how a transmission electron microscope (TEM) works. [3 marks]

Answer 16

1) Pass 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 photographic plate using magnetic lenses.

Electrons pass through the object to create an image on the screen below (the denser the sample, the darker it appears)

Question 17

Describe how a scanning electron microscope (SEM) works. [3 marks]

Answer 17

1) Focus 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.

(It’s like ultrasound)

Question 18

What are the resolutions on an optical, TEM and SEM microscope?

Answer 18

Optical = 0.2 μm

TEM = 0.1 nm

SEM = 20 nm

Question 19

What are the advantages and disadvantages of TEMs? [4 marks]

Answer 19

+ Highest resolution.
+ Higher magnification than optical.

• Whole system must be in a VACUUM - therefore living specimens can’t be observed.
• Complex staining process required & image is still not in colour.
• Image may contain ARTEFACTS
• Specimen must be extremely thin
• 2D image

Question 20

What are the advantages and disadvantages of SEMs? [4 marks]

Answer 20

+ Higher resolution compared to optical microscopes.
+ Higher magnification.

• Whole system must be in a VACUUM - therefore living specimens can’t be observed.
• Complex staining process required & image is still not in colour.
• Image may contain ARTEFACTS

+ Specimen does not need to be thin.
+ 3D image
- Only shows outer surface of specimen.

Question 21

Describe how light microscopes work.

Answer 21

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 the objective lens and eyepiece.