chapter 2 - microscopy

Question 1

what is the total magnification achieved when using the
a) x4 lens
b) x10 lens
c) x 40 lens
on a light microscope

Answer 1

a) x40
b) x100
c) x400

Question 2

what is the process of sectioning and why do we do it when preparing samples?

Answer 2

thin sections of samples are cut, soft sections are often embedded in wax in order to section
why? - because they have to be thin enough for light to pass through them.

Question 3

what is the process of staining and why do we do it when preparing slides?

Answer 3

stains are used which bind to chemicals or structures in or on the cells.
why? enables specimen to be seen under the microscope

Question 4

why might we use differential staining?

Answer 4

some stains bind to specific structures, allowing us to differentiate between the sub-cellular structures

Question 5

what unit is used for cell sizes and larger organelles

Answer 5

micrometres (1000mm)

Question 6

what unit is used for smaller organelle sizes, and molecules

Answer 6

nanometres (1000um)

Question 7

what are the 6 stages of calibrating the eyepiece graticule?
1. ALIGN
2. COUNT
3. NOTE ACTUAL
4. MULTIPLY
5. DIVIDE
6. MEASURE
(NOTE)

Answer 7

1. align the eyepiece graticule with the stage micrometre
2. count the number of divisions on the stage micrometre that the graticule “takes up”
3. note the actual value of one division on the stage micrometre (10um)
4. multiply the value (10um) by the number of divisions the eye piece graticule “took up” - this is the actual length of the graticule
5. divide this by 100 (the number of divisions on the graticule)
6. measure the size of a specimen using the eyepiece graticule
   (NOTE - the graticule has to be calibrated at each objective power)

Question 8

which objective power should you calculate the eyepiece graticule at?

Answer 8

all of them

Question 9

what are the differences between the electron microscopes TEM and SEM

Answer 9

TEM (transmission) electrons pass through specimen
SEM (scanning) electrons bounce off specimen

TEM - image is 2D
SEM - image is 3D

TEM - magnification of up to x500,000
SEM - magnification of up to x100,000

Question 10

what are the resolution limits of
a) a human eye
b) light microscope
c) electron microscope

Answer 10

a) 100um (electrons have a much shorter wavelength than visible light)
b) 200nm
c) 0.1nm

Question 11

what are the benefits of using laser scanning/ confocal microscopes?

Answer 11

high resolution and high contrast
can focus in different depths
multiple images can make a 3D image

Question 12

what profession are laser scanning/ confocal microscopes used in?

Answer 12

medical (specifically to study eye diseases)

Question 13

what are the advantages and disadvantages of light microscopes

Answer 13

advantages:
- portable and safe to use in educational environments
- cheap to purchase and operate
- unaffected by magnetic fields
- living as well as dead material can be viewed

disadvantages:
- only magnifies up to x1500
- the depth of the field is restricted
- limited to 200nm resolution

Question 14

what are the advantages and disadvantages of electron microscopes?

Answer 14

advantages:
- high magnification of SEM - up to 500,000x
- high resolution 0.1nm
- can be used to study any specimen such as animals to metals (organic and inorganic)
- compatible with modern technology

disadvantages:
- costly manufacturing and maintaining
dangerous if left active and unattended
- large piece of floor-to-ceiling equipment
- not portable
- specimen needs to be inside a secure environment such as vacuum chamber (person using it needs to be highly qualified)
- specific requirements to stabilize the specimen and make image as accurate as possible

Question 15

what stain is used for plant cells eg onion
what stain is used for human cells eg cheek

Answer 15

onion - iodine
cheek - methylene blue

Question 16

what do we use to stain DNA, and what colour does it stain?

Answer 16

acetic orcein - stains red

Question 17

what is the formula for working out the surface area and volume of an animal cell (sphere)

Answer 17

SA- 4 x pi x r^2
V - 4/3 x pi x r^3

Question 18

how do you measure the diameter of an animal cell that isn’t a perfect sphere

Answer 18

take 3 or more diameters from different angles and find an average to use to calculate the radius

Question 19

what are the four ways of prepping a sample on a slide?

Answer 19

1. dry mount - solid specimens cut into thin slices, cover slip placed over
2. wet mount - specimen suspended in a liquid before cover slip added
3. squash slides - wet mount prepared then lens tissue used to press down on cover slip
4. smear slide - edge of slide is used to smear the sample creating an even, thin coating

Question 20

what is an ARTEFACT in microscopy?

Answer 20

a visual object or distorted cell structure present in a micrograph, which is not a feature of the specimens, due to sample preparation process,

Question 21

how do laser scanning confocal microscopes produce an image?

Answer 21

focus a laser beam onto a small area on a sample’s surface using objective lenses.
fluorophores in the sample emit photons.
photomultiplier tube amplified the signal onto a detector and an image is produced.