microscopy

Question 1

what is microscopy?

Answer 1

Using microscopes to view objects/specimens that are not visible to the naked eye

Question 2

name the parts of a microscope

Answer 2

• detector
• objective
• specimen
• light conditioning system
• light source

Question 3

where does light travel through the microscope?

Answer 3

goes through the objectives and reaches the detector

Question 4

describe the light microscopic specimen

Answer 4

• cover glass
• sample surrounded by embedding medium (might contain anti-bleaching agent)
• glass slide

Question 5

role of the incubator box?

Answer 5

prevents focus instability

Question 6

how is co2 atmosphere maintained?

Answer 6

controller allows adjustment of air flow and % co2

Question 7

more complexes images…

Answer 7

take longer to capture

If something moves very fast, you need to make sure you capture the sample not the surrounding

Question 8

what is the triangle of frustration?

Answer 8

about signal detection

• temporal resolution
• spatial resolution
• sensitivity

Question 9

pixel area and resolution?

Answer 9

small pixel area = high resolution

Question 10

in order to capture a fast moving image what do you have to sacrifice?

Answer 10

resolution

Question 11

what are some of the markings on objectives and what do they mean?

Answer 11

magnification - how big you can see the sample

application - modify light in order to see different things, some objectives are suitables for certain applications over others

immersion medium – not all objectives can see samples in air, they may need to be immersed in water or a type of oil – e.g. won’t be able to focus if you use on objective that works in air and you immerse it in oil

Numerical aperature - nothing to do with magnification

Question 12

a higher numerical aperture means what?

Answer 12

a better, crispier image of the sample

Question 13

what is meant by light microscopy?

Answer 13

Brightfield: condensing the amount of light that goes through, which gives some 3D properties. Observing a sample

Question 14

3 parts to light microscopy

Answer 14

1) Histology
2) Phase contrast – cell morphology
3) Time-lapse (heart cell differentiation, cell migration)

Question 15

what is histology?

Answer 15

immunohistochemistry
-where is my protein of interest found? find it using antibodies

chemicals that react with different parts of the tissue as they have affinity for basic and acidic components, allowing you to distinguish the different parts of a tissue

PROS: allows you to distinguish the different parts of a tissue

CON: less detail of what the cell is doing

Question 16

what is meant by phase contrast – cell morphology?

Answer 16

a technique used for gaining contrast in a translucent specimen without staining the specimen

Question 17

what is meant by time-lapse?

Answer 17

the method involving live cell imaging from a single observation intime

can show the relevance of movement if for example you find the gene in a virus, remove it and the cells don’t migrate as much without the gene

Question 18

explain what happens in electron microscopy?

Answer 18

2 types:
Transmission EM
Scanning EM

• source of light is a beam of electrons - electrons go through the specimen and find it more difficult to go through certain denser regions and so appear darker
• electrons move faster and more easily through less dense regions, appear lighter

Question 19

what is meant by the fluorescent mode in light microscope?

Answer 19

there is a surge of light, and the light source is a particular wavelength of light
-the light is modified through different filters and eventually reaches the sample, and the sample reflects some type of fluorescence

-transformed to digital data and transmitted to computer

Question 20

flourscence - absorption and emission?

Answer 20

certain types of molecules absorb light – they are excited by a certain wavelength and generate energy (they are at their peak)

at some point the energy is lost (they lose their peak). the excited molecule will then meet another wavelength and reach their peak again (and so on). when they release the energy they emit light

Question 21

what is the stokes shift?

Answer 21

Stokes shift – gain and loss in energy

Different between the wavelength at which the molecule of excited, and the wavelength of emission when the molecule loses its energy

Question 22

what is photobleaching and when does it occur?

Answer 22

bleaching of flurochromes: due to high intensity illumination the flurochromes might lose their ability to permanently emit light

-when gain of energy cannot be released anymore - there’s bleaching. instead of releasing energy and having fluorescence, the fluorescence is lost, and you lose the ability to see the fluroform in detail

Question 23

role of fluorescent proteins?

Answer 23

these proteins can be fused with other proteins and introduced into cells via transection
-this allows the live study of fluorescent tags in organisms/cells

Question 24

confocal vs widespread?

Answer 24

confocal microscopescans a sample with a focused beam of light

only a small volume can be visualised by confocal microscopes at once - bigger volumes need time consuming sampling

Question 25

role of lens?

Answer 25

illuminate and magnify