A2.2 - microscopy theory (3c)

Question 1

how do you calculate magnification?

Answer 1

magnification = eyepiece lens x objective lens

Question 2

how do you calculate image size?

Answer 2

image size = actual size x magnification

Question 3

how do you measure sizes using an eyepiece graticule, including producing a scale bar?

Answer 3

a graticule is located in the eyepiece and is engraved with equal units
- these units are arbitary and the size they measure depends on the particular microscope and the total magnification being used to view the specimen, based on the choice of objective lens
- to calibrate a graticule a stage micrometer is used

a stage micrometer is a slide containing a scale of known size (similar to a tiny ruler)
- by comparing the units of the graticule to the known unit size of the stage micrometer you can determine the size of the object being examined

scale bars are drawn proportionally to a structure of known size, as determined using a graticule and stage micrometer, but with a round-valued length

Question 4

how do you calculate a graticule unit?

Answer 4

comparing the units of the graticule to the known unit size of the stage micrometer allows you to determine the size of the object being examined

Question 5

how do you measure the actual size and image size?

Answer 5

actual size - scale bar
image size - ruler

Question 6

how do you convert between units?

Answer 6

1cm = 10mm = 10000µm = 10000000nm

Question 7

what is the difference between magnification and resolution?

Answer 7

magnification is the increase in an object’s image size compared to its actual size

resolution is the minimal distance between two points at which they have be distinguished as two points

Question 8

how can you identify bacterial cells in a light micrograph?

Answer 8

rod-shaped
stain purple with crystal violet
small (1-10micrometres)

Question 9

how can you identify animal cells in a light micrograph?

Answer 9

nucleus
irregular shape and position
larger (10-50micrometres)

Question 10

how can you identify plant cells in a light micrograph?

Answer 10

nucleus and cell walls
cells are closely packed
chloroplasts
larger (10-100micrometres)

Question 11

how can you identify bacterial cells in electron micrographs?

Answer 11

rod-shaped
flagella may be visible
smaller than eukaryotic cells (1-10μm)

Question 12

how can you identify plant cells in an electron micrograph?

Answer 12

sap vacuole is visible
chloroplast and mitochondria are visible
larger than prokaryotic cells (10-100μm)

Question 13

how can you identify animal cells with an electron micrograph?

Answer 13

mitochondria, but not chloroplasts are visible
larger than prokaryotic cells (10-50μm)

Question 14

what is electron microscopy?

Answer 14

electron microscopes use the interaction of electrons with the specimen to produce an image

Question 15

what are the 2 types of electron microscopy?

Answer 15

scanning electron microscopes (SEM) produce images of the surface of an object

transmission electron microscopes (TEM) use electrons passing through a thin section of a specimen to form an image

Question 16

what are the pros and cons of electron microscopy?

Answer 16

pros:
- higher resolution
- higher magnification

cons:
- specimen must be dead and fixed in plastic
- structural features are produced during preparation which can hinder image accuracy

Question 17

what is light microscopy?

Answer 17

light microscopes use light to form an image with a maximum 2000x magnification, and can use dead or living organisms

Question 18

how are fluorescent stains used in light microscopy?

Answer 18

fluorescent stains bind to specific cellular components and fluoresce when the samples are irradiated with ultraviolet light
- different combinations of dye and cellular component cause different colours of fluoresence

Question 19

how is immunofluorescence used in light microscopy?

Answer 19

immunofluorescence using antibodies with attached fluorescent dyes, so when the antibodies bind to a particular molecule, the dye is located to that area of the cell
- this allows the fluoresence technology to be targeted more precisely that with the standard fluoresence stains
- images fluorescing at different wavelengths can be colorised and merged to study relative locations of proteins within a cell

Question 20

what is freeze fracture?

Answer 20

a way to prepare a sample for observation with an electron microscope; a biological specimen is rapidly frozen, before being physically broken apart (fracturing)
- this process reveals a plane through the sample that can be examined

Question 21

what are the advantages of freeze fracture?

Answer 21

improves our understanding of cell membranes
- the fluid-mosaic model was developed due to micrographs showing proteins in the membrane on both this cytoplasmic and extracellular side, leading to a proposal of two layers of protein on either side of a lipid bilayer

Question 22

what is cryogenic electron microscopy?

Answer 22

allows imaging of single-protein molecules and their interactions with other molecules; one 3D image is formed using computer enhancement of images of many copies of the molecule, which have been frozen in ice

Question 23

what are the advantages of cyrogenic electron microscopy?

Answer 23

enhancing our understanding of virus structure, cell membrane component arrangement, protein synthesis and even gene expression