microscopy

Question 1

describe what a microscope does.

Answer 1

a microscope is an instrument that allows you to magnify objects, making individual cells visible etc.

Question 2

name 4 different types of microscope.

Answer 2

• light microscope
• scanning electron microscope
• transmission electron microscope
• laser scanning confocal microscope

Question 3

what do the abbreviations TEM and SEM stand for?

Answer 3

TEM - transmission electron microscope

SEM - scanning electron microscope

Question 4

outline how an SEM works.

Answer 4

they fire a beam of electrons through a vacuum onto a sample. the electrons are bounced off the sample and detected to produce a 3D image.

Question 5

outline how a TEM works.

Answer 5

fire a beam of electrons through a vacuum onto a sample. the electrons pass through the very thin sample. it passes through dense areas less easily. the electrons are detected underneath the sample to produce a 2D image.

Question 6

outline how a laser scanning confocal microscope works.

Answer 6

a single spot of light is passed across a sample, the dye within the sample causes fluorescence. this is filtered back through a pinhole aperture to the eyepiece.

Question 7

outline how a light microscope works.

Answer 7

it has two lenses, the objective lens near the sample and the eyepiece lens. the two lenses magnify the image. the sample is lit from below .

Question 8

compare the uses of the different microscopes.

Answer 8

light - living or dead sample, staining is used, cheap and most common.
TEM - dead sample. preparation can cause damage. expensive and hard to use. 2D image produced
SEM - similar to TEM but produces 3D images
SLCM - used to view living and moving samples. difficult to stain, provides good contrast.

Question 9

compare the magnification and resolution of the types of microscope.

Answer 9

light - magnification = x2000. resolution = 200nm
SEM - magnification = x500000. resolution = 3-10nm
TEM - magnification = x500000. resolution = 0.5nm

Question 10

define the term resolution

Answer 10

the ability to distinguish two points close together as being separate and to see detail.

Question 11

define the term magnification.

Answer 11

the extent to which an object has been enlarged.

Question 12

state the magnification formula.

Answer 12

magnification = size of image/actual size of object.

Question 13

what is the formula for the overall magnification of an image?

Answer 13

overall magnification = objective lens x eyepiece lens.

Question 14

explain how to convert units when calculating magnification.

Answer 14

1000 nm = 1 um
1000 um = 1 mm
when measurements are getting smaller divide by 1000. when getting bigger multiply by 1000.

Question 15

why is staining useful in light microscopy?

Answer 15

cell structures are often transparent. stains increase contrast as different components of the cell will stain to a different degree. components become more visible and easier to identify.

Question 16

explain how samples are stained in light microscopy.

Answer 16

• first the sample is placed on a slide and allowed to dry.
• it is heat - fixed by passing it through a flame.
• the specimen will stick to the microscope slide and then take up the stain when it is added.

Question 17

name two different stains.

Answer 17

methylene blue stains parts of the cell whereas Congo red stains the background making the cell stand out.

Question 18

describe how to produce a temporary wet mount.

Answer 18

place the cover slip on a 45 degree angle on the edge of the droplet. drop the slip gently. trying to avoid air bubbles.

Question 19

why do slide preparations need to be thin?

Answer 19

so that the layers of cells in the specimen are minimised so individual cells are clearer.

Question 20

what is an eyepiece graticule?

Answer 20

a glass disk marked with a 1 - 100 scale. it has no units and remains unchanged no matter the magnification. the relative sizes of the divisions increases with each increase in magnification.

Question 21

what is a stage micrometer?

Answer 21

a microscope slide with a very accurate scale in micrometres engraves on it. the scale is 100 divisions = 1 mm so 1 division = 10 micrometres.

Question 22

explain how to calibrate a 4 x objective lens..

Answer 22

• put the stage micrometer and eyepiece graticule in place and focus the scale on the micrometer slide.
• align the micrometer scale with the scale in the eyepiece.
• x divisions on the eyepiece graticule = n divisions on the stage micrometer.
• 1 graticule division = number of micrometres/number of graticule divisions.

= magnification factor.