L9: Microscopy

Question 1

What are the parts of a microscope?

Answer 1

→detector
→objective
→specimen
→light conditioning system
→light source

Question 2

What can the light source be?

Answer 2

→halogen

→XBO

Question 3

Describe the light microscopic specimen

Answer 3

→cover glass
→sample surrounded by embedding medium
→glass slide

Question 4

What is the life imaging part of a microscope?

Answer 4

→ the box prevents focus instability

Question 5

How is CO2 atmosphere maintained?

Answer 5

→controller allows adjustment of air flow and %CO2

→air tight table top encloses live cell culture

Question 6

Which cell processes have the shortest experimental time scale?

Answer 6

→microtubules

→cytoskeleton

Question 7

Which cell process have the longest experimental timescales?

Answer 7

→differentiation

→development

Question 8

What is the triangle of frustration?

Answer 8

→user have to find the balance between temporal resolution, spatial resolution, and sensitivity

Question 9

What is temporal resolution?

Answer 9

→low exposure time, low pixel number, binning

Question 10

What is spatial resolution?

Answer 10

→high pixel number, no binning

Question 11

What is sensitivity?

Answer 11

→ high exposure time, binning

Question 12

What are the markings on objectives?

Answer 12

→magnification
→application
→coverslip thickness
→working distance
→immersion medium/numerical aperture

Question 13

What does the aperture of objective determine?

Answer 13

→the resolution

Question 14

What does a high aperture mean?

Answer 14

→The higher the numerical aperture the better the resolution power of the objective.

Question 15

What are the fundamentals of the light microscope?

Answer 15

→Brightfield, DIC, Phase

Question 16

What are the three stages of light microscopy?

Answer 16

1) Histology-
2) Phase contrast
3) Time-lapse

Question 17

How can you locate the protein of interest in histology?

Answer 17

→Using Antibodies

Question 18

What is the role of histology in light microscopy?

Answer 18

→tissue organisation

Question 19

What is the role of phase contrast in LM?

Answer 19

cell morphology

Question 20

What is a problem with using phase contrast in LM?

Answer 20

→Can’t determine how changes in morphology went about

Question 21

What is the role of time-lapse in LM?

Answer 21

→heart cell differentiation, cell migration

Question 22

What are the two types of EM?

Answer 22

Transmission EM

Scanning EM

Question 23

What is the difference between TEM and SEM?

Answer 23

→TEM examine the internal structures of a cell, whereas ascanning electron microscope only allows visualization of the surface of a structure.

Question 24

Why can we use electrons to visualise structures?

Answer 24

Electrons find it difficult to go through high material areas=darker

Question 25

What are the two types of LM set-up?

Answer 25

→bright filed | →fluorescence

Question 26

What are the differences between bright field and fluorescence microscopy?

Answer 26

→Fluorescence= source of light(laser beam) goes through filter cube and emits light source to sample,

Question 27

Why do emitted light shift to longer wavelength?

Answer 27

→due to loss of energy

Question 28

What is photobleaching?

Answer 28

→process whereby fluorophores lose their ability to fluoresce effectively due to high intensity illumination

Question 29

How do you reduce photobleaching?

Answer 29

→use shorter exposure | →reduced excitation light intensities

Question 30

What are fluorescent proteins?

Answer 30

→GFP | → naturally found in light producing cells of cnidarians

Question 31

How are live studies of fluorescent tags carried out in cells?

Answer 31

→fluorescent proteins can be fused with other proteins and introduced in cells via transfection

Question 32

What is the difference between antibodies and protein fusion for fluorescence?

Answer 32

→Antibodies method- cells have to be fixed- when not alive

Question 33

What are the two types of visualising fluoescence?

Answer 33

→antibodies | →protein fusion, tag the gene

Question 34

What are the two types of focuses in microscopy?

Answer 34

→confocal | →widefield

Question 35

What is a difference between confocal and widefield microscopy?

Answer 35

→widefield microscope, the entire focal volume is illuminated, but that creates blur from areas out of focus above and below the image plane →confocal microscope scans a sample with a focused beam of light, higher resolution

Question 36

Compare confocal and widefield microscopy

Answer 36

→confocal has a higher z-resolution and reduced out of focus blur- clearer and crisper → only a small volume can be visualised with confocal

Question 37

What do bigger volumes need in order to be viewed by confocal microscope?

Answer 37

→need time consuming sampling and image reassembling

Question 38

What can be viewed with intracellular live imaging?

Answer 38

→Microtubule dynamics (GFP-tubulin) →Vesicle transport through microtubules (GFP-Kinesin)

Question 39

What are the two types of 3D reconstruction?

Answer 39

→non-neoplastic | →neoplastic