Cells - Methods of Studying Cells

Question 1

What are the equations for magnification?

Answer 1

magnification = image size / actual size

magnification = objective lens x eyepiece lens

Question 2

How do you draw scientific diagrams?

Answer 2

• large
• boundary lines are thin and clear
• use blank paper
• labelling communicates cell features observed
• do not cross label lines
• include magnification or a scale
• most features that are visible are included
• evidence of 3D structure that is visible using controlled focussing
• dotted lines are used for features that are out of the plane of focus
• no sketching, shading, colouring, crossed lines or hanging lines
• use stippling to show darker areas
• if possible, place all labels at the right-hand side of the drawing

Question 3

What is magnification?

Answer 3

The number of times bigger the image/drawing is compared to the object/real size. It is controlled by the power of the lenses used.

Question 4

What is resolution?

Answer 4

The minimum distance needed to differentiate between 2 adjacent objects - it is a measure of the clarity of the image. It is controlled by the wavelength of the illumination used.

Question 5

What is a millimeter?

Answer 5

1x10-3 m

Question 6

What is a micrometer?

Answer 6

1x10-6 m

Question 7

What is a nanometer?

Answer 7

1x10-9 m

Question 8

What is a picometer?

Answer 8

1x10-12 m

Question 9

What is a light microscope?

Answer 9

A type of microscope which has a condenser, objective lens, and eyepiece lens and light is passed through the thin specimen and up through the objective and eyepiece lenses to the eye. Light microscopes use a pair of convex glass lenses that can resolve images that are 0.2um apart. The reason for this is that this is the wavelength of light and therefore restricts the resolution that a light microscope resolves to.

Question 10

What is an electron microscope?

Answer 10

Beams of electrons are used to visualize structures in a vacuum. The vacuum environment is needed so that particles in the air do not deflect/scatter the electrons (which are very small) out of the beam alignment. Electrons have a smaller wavelength than light so electron microscopes have a higher resolution than light microscopes. Electron microscopes can distinguish between items 0.1nm apart. Electrons produce an image when focused onto a fluorescent screen.

Question 11

How do you prepare a temporary mount?

Answer 11

1. Add a drop of water at the centre of the microscopic slide.
2. Cut a thin slice of the sample and lay it at the centre of the microscopic slide (the drop of water will help flatten it).
3. Add a drop of stain to make the sample more visible.
4. Some parts of the sample will absorb more stain than others, producing a black and white image on the screen.
5. Gently lay a microscopic coverslip on top and press it done gently using a needle to remove air bubbles.
6. Touch a blotting paper on one side of the slide to drain excess iodine/water solution.
7. Place the slide on the microscopic stage under low power to observe.

Question 12

What are the differences between a light microscope and an electron microscope?

Answer 12

LIGHT MICROSCOPE

1. use a beam of light
2. use a light bulb which only requires a low voltage
3. use a condenser, objective and eyepiece lens
4. image can be seen directly by the eye
5. the radiation medium that is used is air
6. low magnification (up to x1,500)
7. low resolution (200nm)
8. cheap
9. use glass lenses to focus light rays
10. easy to use
11. can show the structure and arrangement of tissues, microscopic organisms, living or dead, but it cannot show the cell ultrastructure

ELECTRON MICROSCOPE

1. use a beam of electrons which require high voltages for generation
2. use an electron gun
3. use a condenser, objective and eyepiece lens
4. a fluorescent (TV) screen is required to see the image
5. the radiation medium that is used is a vacuum, since electrons are easily scattered by the particles in air
6. high magnification (up to x500,000)
7. high resolution (0.2nm)
8. expensive
9. use electromagnets arranged around the path of the electron beam, which can deflect and thus focus electron beams
10. specialised training required
11. can only show dead structures, but will show the cell ultrastructure including the fine structure of cell organelles

Question 13

What is a scanning electron microscope (SEM)?

Answer 13

All the limitations of the TEM apply to the SEM, except that specimens need not be extremely thin as electrons do not penetrate. In a scanning electron microscope, a beam of electrons is reflected off the prepared surface of the specimen. The sample is covered in gold metal. The beam is then passed back and forth across a portion of the specimen in a regular pattern. The pattern of scattering builds up a 3D image depending on the contours of the specimen. We can build up a 3D image by computer analysis of the pattern of scattered electrons and secondary electrons produced. The basic SEM has a lower resolving power than TEM, but is still better than a light microscope.

• has a lower magnification than TEM
• specimen doesn’t have to be thin
• higher resolving power than light microscope
• can produce 3D image

Question 14

What is a transmission electron microscope (TEM)?

Answer 14

A type of electron microscope that passes a beam of electrons through or past a very thin section of the specimen (which often has been stained with heavy metals to show up the fine internal structures) on their way to the fluorescent screen or photographic film. The beam of electrons is focused onto the specimen by a condenser electromagnet. Areas that absorb the electrons appear darker on the electron micrograph that is produced. Other parts of the specimen allow the electrons to pass through and so appear bright. The image that is produced on the screen can be photographed to give a photomicrograph.

• specimen has to be thin
• higher magnification than SEM
• higher resolving power than light microscope
• produces detailed images

Question 15

What is cell fractionation?

Answer 15

The process where cells are broken up and the different organelles they contain are separated out so that they can be studied in detail.

The most common method of cell fractionation is differential centrifugation.

Question 16

What is homogenation?

Answer 16

The first stage of cell fractionation when cells are broken up by a homogeniser (blender) and organelles are released from the cell. The resultant fluid, known as homogenate, is then filtered to remove any complete cells and large pieces of debris.

Question 17

What is ultracentrifugation?

Answer 17

The second stage of cell fractionation when the fragments in filtered homogenate are separated in a machine called a centrifuge. This spins tubes of homogenate at very high speed in order to create a centrifugal force.

Question 18

What are the limitations of using electron microscopes?

Answer 18

• The whole system must be in a vacuum so living specimens cannot be observed.
• A complex staining process is required and even then the image is not in colour and may introduce artefacts into the image.
• Artefacts are things that result from the way the specimen is prepared. Artefacts may appear on the finished photomicrograph but are not part of the natural specimen. It is therefore not always easy to be sure that what we see on a photomicrograph really exists in that form.
• Specimens have to be very thin, particularly for TEM so that the electrons can pass through. The result is therefore a flat, 2D image. You can partly get over this by taking a series of sections through a specimen. We can then build up a 3D image of the specimen by looking at the series of photomicrographs produced. However, this is a slow and complicated process.
• SEM has a lower resolving power than TEM, but both have greater resolving power than a light microscope.

Question 19

What is the process of cell fractionation?

Answer 19

1. The cells are first blended in an homogeniser forming the resultant fluid called the homogenate. This tube of homogenate is then placed in a centrifuge and spun at a slow speed.
2. The heaviest organelles, the nuclei, are forced to the bottom of the tube where a thin sediment or pellet forms.
3. The fluid at the top, called the supernatant, is removed which leaves just the sediment of the nuclei. The supernatant is then transferred to another tube and spun at a slightly faster speed. This time the pellet that forms contains the next heaviest organelle, the mitochondria, which are forced to the bottom of the tube.
4. This process continues so that each time the speed is increased the next heaviest organelle is sedimented and separated out.

Question 20

What is the limit of resolution?

Answer 20

The limit of resolution is defined as the smallest distance at which it is possible to distinguish two separate items. Up to this point, increasing the magnification will reveal more detail but beyond this point, increasing the magnification will not do this. The object, while appearing larger, will just be more blurred.

Question 21

Under what conditions does cell fractionation occur? Why?

Answer 21

Buffer solution - keeps pH constant so enzymes in the cell’s organelles don’t denature.

Isotonic water potential - prevents water from moving in and out the organelles by osmosis and bursting them (cell lysis) or shrinking them under osmotic pressure.

Cold - so the enzymes work more slowly, preventing destructive enzymes from destroying organelles.

Question 22

What are the relative sizes of the organelles, largest to smallest?

Answer 22

• nucleus
• chloroplasts, mitochondria
• lysosomes, endoplasmic reticulum
• ribosomes

Question 23

Explain how to calibrate and use an eyepiece graticule.

Answer 23

To calibrate a graticule, you observe a special slide called a stage micrometer. When the stage micrometer and graticule are lined up, you can calculate the length that each division on the graticule is equivalent to.

Question 24

How would you prepare cheek cell slides?

Answer 24

1. Using a cotton bud, remove some cells from the inner part of your cheek.
2. Smear them onto a microscopic slide.
3. Add a drop of methylene blue solution to the cells.
4. Carefully place a coverslip over the cells.

Question 25

How would you estimate the area of an onion cell?

Answer 25

1. Place a see-through ruler on the stage.
2. Looking through the eyepiece, estimate the diameter of the field of view.
3. Calculate the area of the field of view.
4. Count the number of cells in the field of view,
5. Divide the area by the number of cells.

Question 26

How would you estimate the length of an onion cell?

Answer 26

1. Place a see-through ruler on the stage.
2. Looking through the eyepiece, estimate the diameter of the field of view.
3. Count the number of cells across the diameter of the field of view.
4. Divide the diameter by the number of cells.

Question 27

How would you calculate the average number of chloroplasts in a moss cell?

Answer 27

1. Count the number of chloroplasts inside 5 cells in a field of view.
2. Repeat in 5 different field.
3. Calculate the average.

Question 28

What should you do if you have difficulty seeing the chloroplasts?

Answer 28

Look at the cells at the edge where the leaf is very thin.

Question 29

What type of light will improve the resolving power of an optical microscope?

Answer 29

Using short wave (bluish) daylight rather than longer wavelength (yellowish) artificial light.

Question 30

Why does an electron microscope have a much greater resolving power than an optical microscope?

Answer 30

Electron beams have wavelengths much shorter than the wavelengths of visible light. The resolving power of a microscope is inversely proportional to the wavelength of the radiation it uses.

Question 31

What skepticism is there about electron microscopy?

Answer 31

The extreme conditions that specimens are put into during preparation, staining and presentation for electron microscopy has led to some skepticism about the technique. The suggestion is sometimes made that some of the features seen using an electron microscope may well be artefacts which are consequences of the preparation technique rather than what is really there in the living cell.

Question 32

What does the condenser control do?

Answer 32

The condenser control focuses the light onto the specimen and produces an image on the objective lens.

Question 33

What does the condenser iris diaphragm do?

Answer 33

The condenser iris diaphragm controls the amount of light entering and leaving the condenser. It is adjusted using a lever. The diaphragm reduces the amount of glare and improves the contrast that can be achieved.

Question 34

What do the coarse and fine controls do?

Answer 34

Focusing adjustments can be made with the coarse and fine controls. These alter the distance between the stage and the lens system. Generally use of the coarse adjustment control is restricted to focusing the low power objective lens.

Question 35

What do the objective and eyepiece lens do?

Answer 35

The objective lens provides the first magnified image. The eyepiece lens magnifies that image and presents it to the viewer.

Question 36

What is a stage micrometer?

Answer 36

A stage micrometer is a slide with a fine scale of known dimension etched onto it.

Question 37

What is a graticule?

Answer 37

A graticule is a fine scale that fits inside an eyepiece lens, and can be used to calculate the size of objects. It can be calibrated using a stage micrometer.

Question 38

What are microscopes?

Answer 38

Microscopes are instruments that produce a magnified image of an object. A simple convex glass lens can act as a magnifying glass but such lenses work more effectively if they are used in pairs in a compound light microscope. The relatively long wavelength of light rays means that a light microscope can only distinguish between two objects if they are 0.2um or further apart. This limitation can be overcome by using beams of electrons rather than beams of light. With their shorter wavelengths, the beam of electrons in the electron microscope can distinguish between two objects only 0.1nm apart.

Question 39

What does the resolving power depend on?

Answer 39

• the wavelength

- form of radiation used

Question 40

What is the speed of centrifugation required for each organelle to be separated out, heaviest to lightest?

Answer 40

nuclei - 1000
chloroplasts - 3500
mitochondria - 3500
lysosomes - 16500
endoplasmic reticulum - 16500
ribosomes - 100000

Question 41

What advances in biological knowledge did cell fractionation and ultracentrifugation enable?

Answer 41

They allowed a detailed study of the structure and function of organelles, by showing what isolated components do.

Question 42

What are the advantages of electron microscopes?

Answer 42

• The electron beam has a very short wavelength and the microscope can therefore resolve objects well (it has a high resolving power).
• As electrons are negatively charged, the beam can be focused using electromagnets.

Question 43

The resolving power of the TEM is 0.1nm. Why cannot this always be achieved in practice?

Answer 43

• Difficulties preparing the specimen limit the resolution that can be achieved.
• A higher energy electron beam is required and this may destroy the specimen.

Question 44

Why must the eyepiece graticule be calibrated?

Answer 44

The scale on the eyepiece graticule cannot be used directly to measure the size of objects under a microscope’s objective lens because each objective lens will magnify to a different degree. The graticule must first be calibrated for a particular objective lens. Once calibrated in this way, the graticule can remain in position for future use, provided the same objective lens is used.