Week 1 - Discoveries, microscopy and fluorescent proteins

Question 1

Types of microscopy in light microscopy?

Answer 1

Bright field
Phase contrast
(DIC) Differential interference contrast
Fluorescent
Confocal

Question 2

What must the cells be in electron microscopy (SEM/TEM)?

Answer 2

Cells must be fixed (killed) to be observable

Question 3

What did Anthoni Van Leeuwenhoek do in 1677?

Answer 3

Described living organisms in pond water too small to visibly see.

Question 4

Who first observed cells? And why did they name them cells?

Answer 4

Robert Hooke - named them after small squarish monk housing called cells.

Question 5

Why are modern microscopes called compound microscopes?

Answer 5

They magnify both the objective (one of three rotating bits that looks at specimen) and the ocular (Bit you look into).
(Compound - two bits in one - objective and ocular).

Question 6

Magnification = image/object

Answer 6

object = image/magnification
Image = magnification * object

Question 7

Why are high power lenses long?

Answer 7

Image is magnified as its projected further from focal point by lens.
(So the further the image takes the focal point (needs to focus on image) from the lens, the more magnified the image becomes).

Question 8

Resolution?

Answer 8

Ability to separate closely spaced points.

Question 9

Cells are colorless and transparent, to be visible they are stained or labelled to improve contrast to cells surroundings. With?

Answer 9

Chemical stains/dyes
Enzyme labels
Fluorescent labels
Electron dense labels

Question 10

Transmitted light contrast modes (ways of observing specimens in light microscopes)?

Answer 10

Brightfield
Phase contrast
Differential Interface Contrast (DIC or Nomarski)

Question 11

Factors of Brightfield, phase contrast and Differential interface contrast (DIC)?

Answer 11

Brightfield - simple setup/ live specimens/ lower magnification, contrast and resolution/ samples have to be stained.
Phase contrast - Can have artifacts/ medium resolution/ needs preparation/ dead specimens/ doesn’t need fluorescence.
Differential interface contrast - live specimens/ dont have to dye specimen/ brilliant resolution/ can be fiddly to observe specimen.

Question 12

What’s chromatic contrast?

Answer 12

Staining the specimen.

Question 13

Examples and colours of specimen staining dye? (chromatic contrast)

Answer 13

(H & E)
Haemotoxylin - stains nuclei blue
Eosin - stains (everything) whole specimen red/pink.

Question 14

Factors of fluorescent microscopy?

Answer 14

• Dark cellular background, fluorescent bright stain labels
• Often in immunochemistry and live cell imaging
• Multiple labelling of more than one colour possible
• Often in confocal microscopy and can be used with specially designed conventional microscopes.

Question 15

What’s a fluorescent molecule?

Answer 15

A Fluorechrome.

Question 16

What makes a molecule a Fluorechrome?

Answer 16

Excite molecule with energy
Electrons jump to next energy level
Then lose energy and return to original energy level
When they do this heat and light are given off/ lost.
This makes it fluorescent as it releases light.

Question 17

Factors of Confocal microscopy?

Answer 17

• Improvement on fluorescent microscopy
• Invented by Harvard student Marin Minsky 1950
• At time they didn’t have existing light detectors so they couldn’t make it a thing.
• For fluorescent use

Question 18

What does Confocal microscopy use?

Answer 18

Lasers of various output wavelengths
Scanning mechanism
Light detectors
Computer with processing power
Suitable fluorochromes.

Question 19

Confocal principle?

Answer 19

The use of a pinhole to exclude out of focus light collected from the microscope refracting from each section of the specimen.

Question 20

Factors of fluorescent proteins?

Answer 20

• Like all other proteins they are continuously synthesized in cells and used in cellular targeting, partitioning and turner processes.
• Bright and non-toxic so cell and tissue life can be manipulated long term.
• Along with sub cellular localization they can be controlled using molecular biological techniques.
• Aren’t stains so remain glowing in cells as permanent markers.

Question 21

Timeline of the discovery and study of the fluorescent protein?

Answer 21

Osamu Shimomura 1962 - Noticed fluorescent protein in the crystal jellyfish.
Douglas Prasher 1992 - Cloned GFP gene but didnt get to test it.
Martin Chalfie 1994 - Expressed the gene in bacteria and it worked.
Roger Tsien 1990-1999 - discovered how to make fluorescent protein colour variants. (Allowed use of protein labels in fluorescent microscopy to use multiple labels of different colours).

Question 22

Factors of (TEM) Transmission electron microscope?

Answer 22

• Make high magnification views of very thin sections of fixed biological tissue.
• Lets us observe extremely thin cross sections of cells
• Black and White.

Question 23

Why are 3D electron microscopes best?

Answer 23

As it lets you section down through imbedded tissue and 3D render them.

Question 24

Factors of (SEM) Scanning electron microscope?

Answer 24

(Same limitations as TEM)
- Difficult to prepare specimen which limits resolution
- Higher energy electron beam required which can destroy specimen.
- Must be in a vacuum so specimen is fixed (dead).
- Can be hard to stain
- Specimen extremely thin
- Image can collect artifacts which obstructs
photomicrographs.