Lab based research methods

Question 1

What are the types of microscope?

Answer 1

1.Stereo :
- reflected light
- can see 3D
2. Compound:
- transmitted light
- requires microscope slide and coverslip (may damage sample)
3 . Inverted:
- transmitted light from above
- objects are below the stahe
- can view samples in a petri dish

Question 2

What is phase contrast microscopy?

Answer 2

• allows live cells to be observed more eaily with no fixation or labelling
• works well with thin speciments

Question 3

What are some advantages of immunohistochemistry?

Answer 3

• straightforward technique
• can be applied to any tissue provided antibody is available

Question 4

Limitations of immunohistochemistry

Answer 4

• limited resolution
• tissue has to be fixed - could lead to artefacts
• need to have an antibody to the protein of interest

Question 5

What are some advantages of phase contrast and DIC microscopy?

Answer 5

• allows live cells to be observed more easily with no fixation or labelling
• relatively inexpensive (compared to other types of microscopes)

Question 6

What are some limitations of Phase contrast and DIC microscopy?

Answer 6

• poor resolution
• cannot identify specific proteins or structures
• 2D images

Question 7

What is fluorescent molecule?

Answer 7

• one that absorbs light of a particcular wavelength and emits light of a different wavelength
• fluorescent stains binf specific molecules:
• DAPI and Hoeschst - bind minor groove of DNA so label nuclei
• phalloidin stains actin
• Fluo3 binds calcium
• can create fluorescent drugs
• intesity of fluorescence can be quantified
• fluorescence decreases with increasinf concentration of antagonist

Question 8

Advantages of fluorescence microscopy

Answer 8

• can identify specific proteins if antibody is available
• can measure concentrations of intracellular signalling molecules/ions e.g. calcium, chloride
• useful in live cells

Question 9

Limitations of florescence microscopy

Answer 9

• prolonged light can cause fading of fluorescence
• not sure whether the prescence of fluorophore can affect intracellular signalling or be toxic to cells
• 2D images

Question 10

What are confocal fluorescence /laser scanning microscopes?

Answer 10

• uses lasers to give specific wavelengths
• confocal microscope = able to focus on a thin layer
• it scans accross the image and then at difffferent depths
• builds up a 3d image

Question 11

What are some examples of multiple fluorescent staining?

Answer 11

• Human cancer cell
• DNA stained blue
• Microtubules are stained red
• protein (INCENP) stained green

Question 12

What is live cell imaging?

Answer 12

If a cell as been transfected with a GFP-linked protein, fluorescence imaging can occcur in live cells
GFP = green fluorescent protein

Question 13

Examples of fluorescent indicator molecules?

Answer 13

• Fluo3-AM
• Fura
• Oregon Green
• Rhod 2
• They fluoresce when the calcium concentration – icnreases and intensity relates to concentration of calcium

Question 14

What is Live vs Fixed imaging?

Answer 14

• cells can be fixed with formaldehyde and then incubated with fluorescently-labelled antibodies
• other fluorescent chemical

Question 15

What are advantages of confocal microscopy?

Answer 15

• can be used for live or fixed cells
• can detect specific proteins or ions/molecules (provided suitable fluorophore available)
• can build 3D image

Question 17

What are some limitations of confocal microscopy?

Answer 17

• restricted to wavelengths avaialable from lasers
• expensive

Question 18

What is electron microscopy?

Answer 18

• uses beams of electrons rather than light
• electron beams have shorter wavelength than light
• have higher resolution -1000000x magnification
• requires a lot of sample preparation (dehydration and embedding within resin then sliced very thinly)