methods of studying cells

Question 1

describe how optical microscopes work.

Answer 1

1. lenses focus rays of light and magnify the view of a thin slice of specimen.
2. different structures absorb different amounts and wavelengths of light.
3. reflected light is transmitted to the observer via the objective lens and eyepiece.

Question 2

outline how a student could prepare a temporary mount of tissue for an optical microscope.

Answer 2

1. obtain thin section of tissue.
2. place plant tissue in a drop of water.
3. stain tissue on a slide to make structures visible.
4. add coverslip using mounted needle at 45 degrees to avoid trapping air bubbles.

Question 3

suggest the advantages of using an optical microscope.

Answer 3

colour image.
can show living structures.
affordable apparatus.

Question 4

suggest the limitations of using an optical microscope.

Answer 4

2D image.

lower resolution than electron microscopes due to longer wavelength of light than electrons - small organelles are not visible.

lower magnification.

Question 5

describe how a transmission electron microscope works.

Answer 5

1. extremely thin specimens are stained and placed in a vacuum.
2. electron gun produces a high energy beam of electrons through the thin slice of specimen.
3. more dense structures appear darker since they absorb more electrons.
4. focus image onto florescent screen using magnetic lenses.
5. image produced is 2D and shows detailed image on internal structure of cells.

Question 6

suggest the advantages of using a TEM.

Answer 6

electrons have a shorter wavelength than light - higher resolution than optical microscope. can see smaller organelles.

higher magnification than optical.

Question 7

suggest the limitations of using a TEM.

Answer 7

2D image.

requires a vacuum - cannot show living structures.

black and white images produced - no colour.

Question 8

describe how a scanning electron microscope works.

Answer 8

1. focus a beam of electrons onto a specimens surface using electromagnets.
2. electrons are scattered in different ways depending on contours.
3. reflected electrons hit a collecting device and are amplified to produce an image on a photographic plate.

Question 9

suggest the advantages of using a SEM.

Answer 9

3D image.

electrons have a shorter wavelength than light - higher resolution than optical.

Question 10

suggest the limitations of using a SEM.

Answer 10

requires a vacuum - cannot show living structures.

black and white images - no colour.

only shows outer surface.

Question 11

define magnification.

Answer 11

how many times larger the image size is than the actual size of the specimen.

Question 12

explain how to use an eyepiece graticule and stage micrometer to measure the size of a structure.

Answer 12

1. line up micrometer on stage to calibrate eyepiece graticule.
2. count how many graticule divisions fit into one division of the micrometer.
3. each division on the micrometer is 10 micrometres, so this can be used to calculate what one division on the eyepiece is at the current magnification.
4. use calibrated values to calculate actual length of structures.

Question 13

define resolution.

Answer 13

minimum distance between two objects in which they can still be viewed as seperate.

Question 14

state an equation to calculate the actual size of a structure from microscopy.

Answer 14

actual size = image size / magnification.

Question 15

outline what happens during cell fractionation.

Answer 15

1. homogenisation. cells are blended in the cold, isotonic, buffered solution to break them open and release organelles.
2. filter homogenate to remove large debris.
3. perform differential centrifugation.

Question 16

outline what happens during differential centrifugation.

Answer 16

1. spin homogenate at high speed in centrifuge.
2. the most dense organelles in the mixture form pellets at the bottom of the tube.
3. filter off the supernatant and spin again, repeating at increasingly higher speeds.

Question 17

what is a pellet?

Answer 17

an isolated organelle.

Question 18

state the order of sedimentation of organelles during differential centrifugation.

Answer 18

most dense - least dense.

nucleus - chloroplasts - mitochondria - lysosomes - rough endoplasmic reticulum - plasma membrane - smooth endoplasmic reticulum - ribosomes.

Question 19

explain why fractionated cells are kept in a cold, buffered, isotonic solution.

Answer 19

cold - to reduce enzyme activity. when cell is broken open enzymes are released which could damage organelles.

buffered - maintain constant pH to prevent damage to organelles.

isotonic - organelles must be the same water potential as the solution to prevent osmosis, as this could cause organelles to shrivel and burst.