Questions on microscopy, magnification and resolution

Question 1

An eyepiece graticule is calibrated against a stage micrometer whose divisions are each 10 µm long. 25 eyepiece divisions are found to cover 4 stage micrometer divisions. A cell then measures 15 eyepiece divisions across. Calculate the diameter of that cell in micrometres.

Accepted Answer

M1 4 stage micrometer divisions correspond to 4 × 10 = 40 µm M1 one eyepiece division is worth 40 divided by 25 M1 = 1.6 µm per eyepiece division, then multiplied by the 15 divisions the cell covers A1 a final answer of 24 µm

Question 2

Explain why a transmission electron microscope resolves finer detail than a light microscope, and explain why it cannot be used to look at a living cell.

Accepted Answer

B1 resolution is limited by the wavelength of the radiation used to illuminate the specimen B1 a beam of electrons has a far shorter wavelength than visible light, so points much closer together can still be seen as separate B1 the electron beam has to travel through a vacuum, because air molecules deflect electrons and scatter the beam B1 a vacuum removes the water from a cell, so the specimen must be dead, fixed and dehydrated before it is put in

Question 3

Compare a transmission electron microscope with a scanning electron microscope, referring to the specimen used, the electrons detected, the image produced and the resolution achieved.

Accepted Answer

B1 a transmission instrument needs an ultrathin section, whereas a scanning instrument takes a whole specimen coated in a thin film of metal B1 a transmission instrument detects electrons that have passed through the specimen, whereas a scanning instrument detects electrons knocked off its surface B1 a transmission instrument gives a two-dimensional image of a section, whereas a scanning instrument gives a three-dimensional image of a surface B1 a transmission instrument resolves to about 0.1 nm, which is better than the few nanometres a scanning instrument manages

Question 4

The scale bar under the mitochondrion represents 1 µm. Calculate the actual length of the mitochondrion. On a printed copy of the same micrograph the scale bar measures 15 mm; calculate the magnification of that printed copy.

Accepted Answer

M1 the organelle is three bar lengths long, measured against the bar itself A1 3 × 1 µm = 3 µm, which is a reasonable length for a mitochondrion M1 magnification = size of image ÷ actual size, working in one unit: 15 mm ÷ 0.001 mm A1 × 15 000, with no unit, because a magnification is a ratio

Question 5

Describe how ultracentrifugation separates organelles, and describe the order in which nuclei, mitochondria and ribosomes are pelleted.

Accepted Answer

B1 the tube is spun, and the largest and densest contents travel outwards fastest and collect as a pellet at the bottom, with everything still suspended left in the supernatant B1 the supernatant is decanted into a fresh tube and spun again at a higher speed, so the next heaviest component pellets in its turn B1 the organelles pellet in order of decreasing size and density: nuclei first, then mitochondria B1 ribosomes pellet last of all, because they are the smallest structures collected, requiring the highest speeds

Question 6

Describe the stages a specimen goes through in preparation for viewing with a transmission electron microscope, from fresh tissue to a stained section.

Accepted Answer

B1 the tissue is fixed in chemicals, to preserve its structure B1 it is dehydrated through a series of solvents, removing the water B1 it is embedded in resin and cut into ultrathin sections B1 the sections are stained with salts of heavy metals, such as lead or osmium, to give contrast

Question 7

A ribosome measures 5 mm across on an electron micrograph labelled as being at a magnification of 200 000 times. Calculate the actual diameter of the ribosome, giving your answer in nanometres.

Accepted Answer

M1 actual size = image size divided by magnification M1 converts 5 mm to 5 000 000 nm before dividing, or works consistently in another single unit A1 an answer of 25 nm, which is the expected size for a ribosome

Question 8

A student examining an electron micrograph counts what appear to be forty separate mitochondria, of widely differing sizes and shapes, in one small cell. Suggest two reasons why this count and these shapes may be misleading.

Accepted Answer

B1 the micrograph is one thin section through a three-dimensional cell, so each organelle is seen only where the knife happened to cut it B1 a mitochondrion cut across appears as a small circle and one cut along its length as a long sausage, so the same organelle gives different profiles B1 a bent organelle can be caught by the knife twice, so one mitochondrion appears as two separate profiles and the number is overcounted

Question 9

Explain why the solution used to homogenise tissue in cell fractionation must be cold, isotonic and buffered.

Accepted Answer

B1 cold: this slows the action of hydrolytic enzymes, such as those from lysosomes, that would otherwise digest the organelles being collected B1 isotonic: this prevents water moving in or out of the organelles by osmosis, so they do not burst or shrivel B1 buffered: this keeps the pH constant, so proteins and enzymes retain the shape their function depends on

Question 10

A student enlarges a photograph taken through a light microscope until it is a metre wide, and claims this has increased the magnification enough to reveal detail invisible in the original image. Explain why this claim describes empty magnification.

Accepted Answer

B1 magnifying an image without limit does not add detail beyond what the resolution of the original instrument recorded B1 resolution is fixed by the wavelength of the radiation used to form the original image, not by any later enlargement of it B1 the enlargement produces a bigger but equally blurred image, containing no more detail than the original, which is what empty magnification means

Question 11

The mesosome, an inward fold of the bacterial cell membrane, was seen in early electron micrographs prepared by one particular fixation method, but not when a different fixation method was used on the same species. Suggest what this comparison shows about the mesosome, and suggest how biologists in general decide whether a structure seen in one preparation is real or an artefact.

Accepted Answer

B1 the mesosome is probably an artefact produced by that particular fixation chemical rather than a structure present in the living bacterium B1 a structure seen after several different preparation methods is likely to be real, because it is unlikely that different chemicals would each create the same appearance by accident B1 a structure seen with only one particular technique is probably an artefact of that technique

Question 12

Compare the images produced by a light microscope with those produced by a transmission electron microscope, referring to colour, whether a living specimen can be examined, and resolution.

Accepted Answer

B1 a light microscope image is in colour, natural or from stains, whereas a TEM image is black and white, with any colour added afterwards B1 a light microscope can show a living, moving specimen, whereas a TEM specimen is dead, because it is fixed, dehydrated and examined in a vacuum B1 a light microscope resolves to about 0.2 µm against about 0.1 nm for a TEM, so a TEM reveals internal ultrastructure that is invisible with light

Question 13

State what is meant by the magnification of an image, and state what is meant by the resolution of a microscope.

Accepted Answer

B1 magnification is how many times larger the image is than the actual object, and is a ratio with no units B1 resolution is the minimum distance between two points at which they can still be distinguished as separate

Question 14

A researcher separating organelles by ultracentrifugation obtains a mitochondrial pellet that also contains lysosomes. Explain why this pellet is not pure mitochondria.

Accepted Answer

B1 lysosomes and mitochondria are similar in size and density, so they sediment together at the same centrifuge speed B1 a pellet is therefore enriched in the organelle of interest but never completely pure

Question 15

Explain why an eyepiece graticule must be calibrated separately for each objective lens used on a microscope.

Accepted Answer

B1 the objective lens magnifies the image of the stage micrometer, but it does not magnify the eyepiece graticule's own divisions, which stay the same apparent size B1 so each objective gives an eyepiece division a different real value in micrometres, and a calibration made for one lens is not valid for another