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2.1.1 microscopy Flashcards

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1
Q

cell theory

A
  • plant and animal tissues are made up of cells
    -cells are the basic unit of all life
  • cells only develop from existing cells
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2
Q

magnification

A

how many times bigger an image appears, compared to the real
specimen

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3
Q

resolution

A

the ability of a microscope to distinguish between 2 points that are
close together. The higher the resolution, the clearer the image,
the more detail can be seen

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4
Q

what is a high resolution?

A

a clearer image can be seen, meaning there is more detail seen too

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5
Q

how many lenses does a compound light microscope have

A

2

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6
Q

name the 2 lenses a light microscope has

A

objective lens
eyepiece lens

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7
Q

what does the objective lens do?

A

it is placed near the specimen and produces a magnified image

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8
Q

what does the eyepiece lens do?

A

it is what the specimen is viewed through and it magnifies the image produced by the objective lens

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9
Q

what does the objective/eyepiece configuration in a light microscope allow for?

A
  • higher magnification
  • reduced chromatic aberration than in a simple light microscope
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10
Q

how is illumination provided in light microscopes?

A

by a light underneath the sample

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11
Q

how can you calculate the total magnification in a light microscope?

A

total = eyepiece mag x objective mag

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12
Q

how do you prepare a microscope slide?

A
  • use a temporary mount (specimen is suspended in liquid)
    1. pipette small amount of water onto slide
    2. use tweezers to place thin section onto water
    3. stain added to the sample
    4. add cover slip, without getting any air bubbles in there
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13
Q

why do you stain the sample?

A
  • To give CONTRAST to the image as some parts of the sample will take up
    the stain and other parts won’t
  • Makes the image VISIBLE (without a stain, most biological structures
    are transparent, will all look the same)
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14
Q

safety procedures whilst dealing with slide?

A

-carry a microscope with both hands
-store microscopes with the lowest power lens in place
-cover microscope with dust sheet when not in use

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15
Q

fixing

A

Chemicals (e.g. formaldehyde) used to preserve the specimen in as near natural state as possible

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16
Q

sectioning

A

specimen is dehydrated with alcohol, then placed in a wax mould to harden it, then sliced very thinly (thin enough to let light through)

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17
Q

staining

A

Provides contrast as diff parts of the specimen take up diff stains,
giving diff colours, which you can then see

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18
Q

mounting

A

specimens secured to a microscope slide, with a permanent cover-slip on top

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19
Q

RULES FOR DRAWING WHAT YOU OBSERVE WITH A LIGHT MICROSCOPE

A
  1. title
  2. state mag
  3. sharp pencil
  4. use as much paper as possible
  5. smooth, continuous lines
  6. do not shade
  7. clearly defined structures
  8. correct proportions
  9. label lines should not cross or have arrow heads
  10. label lines should be parallel to top of page
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20
Q

laser scanning confocal microscope

A
  1. uses laser beams to scan specimen, which can be tagged with a fluorescent dye, so fluorescent gives off light
  2. light is focused through a pinhole onto a detector, which generates a 3D image
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21
Q

use of pinhole in LSCM

A

means out of focus light is blocked, so microscope can produce clearer image than normal light microscope, increasing resolution

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22
Q

what are LSCMs used for?

A

thick specimens

23
Q

what does the laser in an LSCM do?

A

improves illumination as it gives a higher light intensity

24
Q

what does it mean by LSCM have depth selectivity?

A

they can focus on structures
at different depths within a specimen

25
Q

what is depth selectivity useful for?

A

observing whole living
specimens as well as cells

26
Q

define fluorescence

A

emission of light that has been absorbed

27
Q

limitation of LSCM that cannot be used for deep tissue imaging?

A

penetration of sample is limited

28
Q

cm to mm

A

x10

29
Q

mm to μm (micrometres)

A

x1000

30
Q

μm to nm

A

x1000

31
Q

what happens if resolution is poor?

A

two objects that are very close together will be seen as one

32
Q

why do light microscopes not have good resolution? (4 points)

A
  1. wavelength of light is long.
  2. Light waves get reflected from structures in the specimen as they pass through it. These waves then get diffracted (spread out) so that they end up overlapping with each other.
  3. this means structures from where this light came are no longer seen
    as being separate to each other and the detail is lost.
  4. the best resolution is 200nm
  5. this also affects the mag
33
Q

how can you calibrate a microscope so the specimen can be measured?

A
  • eyepiece graticule has a scale, with no units
  • stage micrometer is viewed
  • line them up
  • find out what each EPG division’s unit is
34
Q

if you want to look at the specimen using the different objective lenses what do you have to do and why?

A

re-calibrate as on each magnification , the proportion of the stage micrometer visible changes

35
Q

how does a transmission electron microscope work?

A
  • a beam of electrons is fired at the specimen
  • a series of electromagnets focuses the electrons on to the specimen
  • denser parts of the specimen absorb electrons (stop them passing through) and less dense areas let the electrons through
  • the electrons are focused on to a screen which detects them, and an image is produced.
36
Q

type of image produced by a TEM

A

2D, black and white, shows internal detail of specimen

37
Q

why is a beam of electrons being used better than light?

A

the wavelength of light is shorter than of the beam, so you get a higher higher resolution and mag in a electron microscope

38
Q

limitations of a TEM and SEM

A
  1. specimens need to be sliced very thinly to allow electrons through
  2. needs to be a vacuum inside as any air molecules would deflect the electron beam
  3. so only dead specimens can be viewed
39
Q

The specimen sits on a copper grid rather than a glass slide, why?

A
  1. A glass slide would stop the flow of electrons
  2. The electrons would not reach the screen
  3. Copper grid has holes in it to let electrons through
40
Q

how does a scanning electron microscope work?

A

scan a beam of electrons across the surface of a specimen, and any
reflected electrons are collected.

41
Q

what type of image is produced by a SEM?

A

3D surface image of the specimen

42
Q

The advantage of the SEM compared to TEM

A

the specimens do not need to be cut
quite as thin, since the electrons do not need to pass through

43
Q

disadvantage of the SEM compares to the TEM

A

the images are lower
magnification and resolution

44
Q

staining in electron microscopy

A

solutions of heavy metal salts that attach to some parts of the specimen and not others. heavy metal ions scatter the electrons that are fired at the sample, giving contrast between structures.

45
Q

9 differences between light and electron microscopes

A

Light
1. inexpensive to buy
2. small and portable
3. simple sample preparation
4. vacuum not required
5. can view living or dead samples
6. natural colour can be seen
7. coloured stains/dyes used for contrast
8. Max. magnification X2000
9. Max resolution 200nm (0.2µm)

Electron
1. expensive
2. large, needs to be installed
3. complex sample prep
4. needs vacuum inside
5. can only view dead sample
6. black and white images
7. Heavy metal salts used for contrast
(“false colour” can be added digitally)
8. Max magnification;
X 1000 000 for TEM
X 500 000 for SEM
9. Max resolution;
0.5nm for TEM
3-10nm for SEM

46
Q

max mag of light microscope

A

x2000

47
Q

max resolution of light microscope

A

200nm (0.2µm)

48
Q

max mag of TEM

A

x 1,000,000

49
Q

max mag of SEM

A

x500,000

50
Q

max resolution of TEM

A

0.5nm

51
Q

max resolution of SEM

A

3-10nm

52
Q

what are artefacts?

A

These are visible structures on the image which are not really part of the specimen;
e.g. air bubble when preparing a slide for a light microscope
specks of dust when preparing specimens for an electron microscope

53
Q

where are artefacts most likely to occur?

A

electron microscope because the sample preparation is a lot more complicated

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