L19: Molecular/Cellular Imaging

Question 1

What is the principal of optical magnification?

Answer 1

A microscope generates a magnified image of a define object

Question 2

What are the basic components of a compound light microscope using transmitted light as illumination?

Answer 2

• Tungsten lamp produces white light, collimated by a condenser
• the light trans-illuminates sample on the stage
• the objective lens is the ‘heart piece’ of the microscope, collecting the light emitted from the sample and providing the major magnification of th eimage
• the eyepiece provides a second step of magnification and projects the magnified image onto the retina

Question 3

How are use of histological dyes and bright field microscopy useful for a wide range of basic tissue analysis?

Answer 3

Brightfield micrographs of fixed and stained histological specimens: Haematoxylin (DNA/blue) and Eosin (proteins/red) - standard histology dyes

Question 4

What are the disadvantages of histological dyes and bright field microscopy?

Answer 4

• lack of specificity, e.g. labelling specific compounds (DNA and proteins - wide range)
• low contrast images

Question 5

How is fluroescence microscopy an improvement in optical imaging?

Answer 5

Improvement of specificity and contrast methods alllows the targeted labelling of cells, organelles and molecules

Question 6

What is the principle of exploitation of fluorescence for high contrast optical labels?

Answer 6

Electrons lifter to higher orbital, when drops back to the orbital, emits the photon, thus fluorescence

Question 7

What is the basic principal of fluorescence microscopy?

Answer 7

• Fluorescence microscopy allows the selective and specific detection of molecules which can be made to fluoresce: naturally (autofluoresce) or labelled with fluorophores (secondary fluorescence)
• irradiate the specimen with excitation light
• then visualise the much weaker emission light (without visualising the excitation light)

Question 8

What are the different types of fluorescent labels?

Answer 8

• synthetic fluorophores
• fluorescent antibody conjugates
• fluorescent fusion proteins

Question 9

What are the advantages and disadvantages of using synthetic fluorophores?

Answer 9

Advantages: easy to apply, bright fluorescence, independent from protein expression, highly functional
disadvantages: limited specificity, difficult to target specific molexules (tubulin - not specific enough)

Question 10

How does fluorescent immunohistochemistry work?

Answer 10

Fluorescent molecules covalently bound to antibodies.
Two types:
- direct: primary antibody conjugates to the fluorescent probe –> fast and highly specific but no signal amplification and little flexibility in fluorophore/colour combinations
- indirect: two-step process –> can amplify the signal and allows for flexible combinations of primary and secondary antibodies

Question 11

What is the main advantage of fluorescent labels for in situ labelling?

Answer 11

Looks at live specimen, allows direct in situ labelling using expression of fluorescent proteins, essential for live cell experiments;
many variants, colour-shifter, pH-sensitive

Question 12

What are the pros and cons of using fluorescent fusion proteins as markers?

Answer 12

cons - problems caused by over-expression, expression on top of endogenous protein, sterical hindrance, oligomerisation
pros- highly specific, can be used as label or reporter

Question 13

What’s different in microscope set up in live cell imaging?

Answer 13

has a chamber that controls:
- temperature
- gas
- humidity
- light

Question 14

What is an example for functional imaging: fluorescence recovery after photobleaching (FRAP)?

Answer 14

FRAP to quantify the movement of specific molecules.
Live specimen assay to study the mobility of fluorescence-labelled cell components.
Photo-bleach region of interest (ROI) - destroys all local fluorescence.
Track ‘fluorescence repopulation’ of bleached are over time.
Then a graph is made and dynamics can be quantified.