Microscopy

Question 1

What are the three principles of cell theory?

Answer 1

1) All living organisms are composed of one or more cells
2) The cell is the basic unit of structure and organisation in organisms
3) Cells arise from pre-existing cells

Question 2

What did Robert Hooke contribute to cell theory?

Answer 2

• He observed cells in cork
• Was the first man to use the word ‘cell’
• Wrote a journal of his micro graphs called micrographia.

Question 3

What did Antonie van Leeuwenhoek contribute to cell theory?

Answer 3

• First person to document the microscopic observations on LIVE samples
• Such as bacteria, red blood cells, spermatozoa, muscle fibres
• Called these microbes ‘tiny animals’
• Found that living things were indeed made up of cells.

Question 4

What did Barthélemy Dumortier contribute to cell theory?

Answer 4

• First person to observe cell division in plants using microscopy
• This was used as evidence for cell theory, that cells were created from old ones, rather than spontaneously created.

Question 5

What did Theodor Schwann contribute to cell theory?

Answer 5

-Proposed the extension of cell theory from just plants to animals as well.

Question 6

What did Robert Remak contribute to cell theory?

Answer 6

• Discovered that the origin of cells was by the division of pre existing cells
• Observed cell division occur in frogs eggs immediately after fertilisation.

Question 7

What did Louis Pasteur contribute to cell theory?

Answer 7

• Had many discoveries in vaccinations, microbial fermentation and pasteurisation.
• FERMENTATION provided firm evidence against the spontaneous generation of life and cells
• Allowed full acceptance of full cell theory.

Question 8

What was Louis Pasteur’s pasteurisation experiment?

Answer 8

• Heated three swan neck flasks
• One was left open and no bacteria was present
• Bacteria couldn’t reach fermentable liquid, which went against spontaneous creation
• One had the long neck removed
• Bacteria could reach fermentable liquid
• One was tipped to the side
• Bacteria could reach fermentable liquid.

Question 9

What are the disadvantages of electron microscopy?

Answer 9

• Expensive to buy and operate
• Specimens have to be dead
• Complex sample preparation

Question 10

How does a light microscope work?

Answer 10

• Light is focused by glass lenses
• Light is passed through specimen
• Light absorbed creates the image
• Image is magnified by objective lens, then again by eyepiece lens.

Question 11

What are the advantages of a light microscope?

Answer 11

• Inexpensive to buy and operate
• Don’t require a lot of training to use
• Vacuum isn’t required (can use on live samples)
• Simple sample preparation.

Question 12

What are the disadvantages of a light microscope?

Answer 12

• Relatively low resolution 200nm

- Relatively low magnification 2000x

Question 13

What is the equation for magnification?

Answer 13

Magnification=Image
_____
Actual

Question 14

One eyepiece graticule division equation

Answer 14

Number of micrometers on the slide micrometer
______________________________________
Number of eyepiece graticule divisions.

Question 15

How to prepare a dry mount microscope slide?

Answer 15

• Solid specimen viewed whole or cut into thin slices
• Specimen is place on centre of slide
• Cover slip placed over specimen
• Used for pollen, insects, etc.

Question 16

How to prepare a wet mount slide?

Answer 16

• Specimen is suspended in liquid such as water or immersion oil
• Cover slip placed over specimen at an angle (avoid air bubbles)
• Aquatic samples can be viewed this way

Question 17

How to prepare a squash slide?

Answer 17

• Prepare a wet mount slide
• Use lens tissue to gently push down on cover slip to squash sample
• Can also squash sample between to microscope slides as cover slip is delicate
• This can be used for root tip squashes to observe cell division.

Question 18

How to prepare a smear slide?

Answer 18

• Edge of microscope slide used to smear sample onto another microscope slide
• This creates a thin, even coating
• Cover slip is place over sample
• Used on samples of blood to look at cells.

Question 19

Why must specimens be thin?

Answer 19

To allow light to pass through

Question 20

Why must the refractive index of medium used in wet mount be the same as glass?

Answer 20

Because this will keep the rays of light straight so an in-focus image is produced.

Question 21

How to focus a microscope?

Answer 21

• Move stage to the highest level using coarse focus mechanism
• Start with the lowest magnification objective lens
• Place your microscope slide on stage, held in place by stage clips
• Move the stage down until it comes into view and focus using the fine focus mechanism.

Question 22

Why do we need to calibrate microscopes?

Answer 22

Because the magnification stated on each lens can vary, so we need to calibrate so we can find the true magnification.

Question 23

Why do we stain specimens?

Answer 23

• Higher contrast because different organelles take up more stain than others
• More internal structure visible

Question 24

What are the rules for a good scientific drawing?

Answer 24

• Drawing and label lines done in sharp pencil
• Drawing should take up at least half the page
• Lines need to be continuous
• Ensure proportions are correct
• Add labels of what you see
• Add extra ANNOTATIONS (colour, texture)
• State magnification
• Have a title and name stain used
• Make sure label lines touch what you’re labelling
• NO shading or colour

Question 25

What are the differences between the eyepiece graticule and stage micrometer?

Answer 25

- Eyepiece graticule remains constant regardless of the magnifications, whereas the stage micrometer increases in size - This way, you can calibrate the microscope at every magnification.

Question 26

How does a transmission electron microscope work?

Answer 26

- Beam of electrons, in a vacuum, are transmitted through a sample from an electron gun - The beam is focused by electromagnetic lenses - Beam passes through specimen and either scatter or hit a fluorescent screen at the base of the microscope, which magnifies the image.

Question 27

How are samples prepared for TEM microscopy?

Answer 27

Samples are embedded in resin, stained with heavy metal compounds and cut into into ultra thin sections.

Question 28

How does a scanning electron microscope work?

Answer 28

- A beam of electrons, in a vacuum are focused by electromagnetic lenses - The fine beam of electrons scans the surface - The secondary electrons emitted by atoms which have interacted with the electron beams are detected using secondary electron detector. - These secondary electrons that are collected, create the image.

Question 29

What are the advantages of electron microscopy?

Answer 29

- Much higher resolution - Allow a cell's ultrastructure to be seen - Resolving power of TEM 0.5nm - Resolving power of SEM 3-10nm - Higher magnification over 50,000X - TEM produces detailed images of cell ultrastructure - SEM produces 3D images and great surface detail of an object inside a cell

Question 30

How are samples prepared for SEM microscopy?

Answer 30

- Have to be small enough to fit on the specimen stage - Needs special preparation to increase sample's electrical conductivity - Non-conduction specimens are coated with an ultra thin layer of gold, platinum, chromium, etc.

Question 31

What are artifacts?

Answer 31

Anomalies apparent in both microscopy and photography.

Question 32

What is an example of an artefact?

Answer 32

Mesosomes - People believe they were part of a cell’s ultrastructure but they actually don’t exist - They in fact were artefacts

Question 33

What are the disadvantages of electron microscopes?

Answer 33

- Expensive to buy and operate - Compex sample preparation - Vacuum is required - Specimens have to be dead

Question 34

How does a fluorescence microscope work?

Answer 34

- Uses fluorescence to create an image of a specimen - Fluorescent dyes contain fluorophores - Fluorophores emit longer wavelengths of light - This creates a high resolution image - The dichroic mirror reflects only one colour of light which passes through the specimen.

Question 35

How does a laser scanning confocal microscope work?

Answer 35

- Is a refinement of the fluorescence microscope - Single spot of focused light focused over specimen and causes fluorophores in sample to fluoresce - A confocal pinhole blocks any out of focus light, and therefore the image is higher resolution.

Question 36

How does a super resolved fluorescent microscope work?

Answer 36

It creates a higher detail image by combining many smaller and overlapping images of a specimen AND Digitally superimposing many images on top of each other to create a higher resolution image. RESOLUTION BREAKS THEORETICAL LIMIT OF 0.2nm for optical microscopes

Question 37

How does an atomic force microscope work?

Answer 37

Microscope generates images by scanning a cantilever over the surface of a sample. Can generate images of things the size of fractions of a nanometer -1000x more than optical limit -Shape traced with cantilever is processed and an image is created.

Question 38

What is the definition of magnification?

Answer 38

How many times larger the image is than the actual size of the object being viewed

Question 39

What is the definition of resolution?

Answer 39

Ability to see individual objects as separate entities

Question 40

How to convert mm to nm?

Answer 40

1x10^6

Question 41

How to convert mm to um?

Answer 41

1x10^3