Chapter 1: Microscopy

Question 1

What is used to produce ultra-thin slices of specimen to observe under a microscope? what are these clices called?

Answer 1

A microtome.

Sections.

Question 2

What was the theory of spontaneous generation? Why was it proposed and believed?

Answer 2

That life could be produced from non living matter. Proposed from the existence of life in seemingly sterile conditions.

Question 3

How was spontaneous generation disproved?

Answer 3

Louis Pasteur’s swan-neck experiment, demonstrated how bacteria only grew in sterile conditions after being exposed to air (which contained bacteria)

Question 4

how do light microscopes work?

Answer 4

light from a mirror is reflected through a specimen into the objective lens, which magnifies it. hen it is further magnified by the eyepiece lens

Question 5

advantage of compound microscopes over simple ones?

Answer 5

reduced chromatic aberration/distortion

Question 6

advantages of light microscopes?

Answer 6

• can observe living specimens
• no harsh chemicals involved
• easy to set up
• inexpensive
• can see in colour or if using a stain
• vacuum not required
• sample preparation doesn’t lead to distortion

Question 7

disadvantages of light microscopes?

Answer 7

• low magnification
• low resolution
• cannot see ribosomes

Question 8

magnification/resolution of LM

Answer 8

M = 2000
R = 200 nm

Question 9

how does a laser scanning confocal microscope work?

Answer 9

a single spot of laser light of high energy illuminates chemical dyes on a specimen, causing them to fluoresce.

Question 10

what is fluorescence?

Answer 10

absorption and re-radiation of light to a longer wavelength and lower energy / thus fluoresce

Question 11

advantages of LSCM

Answer 11

• high depth selectivity and so can veiw whole cells
• can see living specimens
  can track molecules
• high secolution than LM

Question 12

disadvantages of LSCM?

Answer 12

• more expensive

- more complex

Question 13

how can we get 3D images from LSCM?

Answer 13

by using light from more than one focal point

Question 14

why is a laser used instead of light in LSCM?

Answer 14

higher intensities, improves illumination, and higher energy, so light re-emmitted is fluorescent

Question 15

define the term relsolution (microscopy)

Answer 15

the ability of a microscope to distinguish between two points on a specimen / the smallest interval measurable by a microscope

Question 16

define magnification

Answer 16

how much an image can be magnified from the original

Question 17

uses of LSCM?

Answer 17

• used to observe fungal filaments with in the cornea
• new drugs
• virtual biopsies

Question 18

how does a TEM work?

Answer 18

• beam of electrons fired from cathode and focused using magnets
• ransmitted through a specimen and focused
• form image

Question 19

advantages of TEM?

Answer 19

• can see details inside cells

Question 20

disadvantages of TEM?

Answer 20

• general EM dis

Question 21

magnification/resolution fo TEM

Answer 21

M = 500,000
R = 0.5 nm

Question 22

how does a SEM work?

Answer 22

• beam of electrons fired form cathode and focused by magnets
• electrons sent across surface of specimen
• secondary electrons reflected and collected

Question 23

advantages of SEM

Answer 23

• 3D images produced

Question 24

disadvantages of SEM

Answer 24

• specimen must be dehydrated and metallic salt stains placed
• sample preparation causes distortion
• lower resolution

Question 25

magnification/resolution of SEM

Answer 25

``` M = 200,000 R = 3-10 nm ```

Question 26

general disadvantages of EM ?

Answer 26

- grayscale images, digital false colour can be added - generally dead - expensive - complex, need skill/training - vacuum required - sample preparation may cause distortion of image -

Question 27

magnification equaiton?

Answer 27

M=I/A

Question 28

purpose of differential staining?

Answer 28

- different organelles absorb different amounts of stain | - thus we can differentiate between them

Question 29

all purpose stain (stains all)

Answer 29

methylene blue

Question 30

stain that binds to DNA and stains chromosomes what colour

Answer 30

- acetic orcein | - dark red

Question 31

stains cytoplasm?

Answer 31

- eosin

Question 32

stains lipids?

Answer 32

- sudan red

Question 33

stains cellulose in plant walls WHAT COLOUR and starch granules WHAT COLOUR

Answer 33

- iodine in potassium iodide - culluose walls yellow - starch granules blue/black: violet under microscope

Question 34

what is done by experts to specimens before use in labs?

Answer 34

- specimens dehydrated - embedded in wax to prevent distortion during slicing - sliced using microtomes (knives) to make VERY THIN slices called SECTIONS - fixing using formaldehyde to stop specimens in near natural state

Question 35

how to you use/calibrate an eyepeice graticule using a stage micrometer?

Answer 35

- clip in stage micrometer - place EPG into eyepeice lens - align, measure from 2 intervals - MATHSSSS

Question 36

types of mounts? | types of slides?

Answer 36

- wet mount - temporary mount - dry mount + smear slide + squash slide

Question 37

how is each mount/slide prepared?

Answer 37

- dry: specimen sliced using microtome - wet: specimen immersed in liquid, cover slip at 45 - temporary mount: in liquid evaporates, cover slip - squash: soft specimen, wet mount squashed using cover slip - smear: edge of cover slip used to smear, dropped at 45