FLUORESCENCE MICROSCOPY

Question 1

WHAT IS FLUORESCENCE?

Answer 1

→ when a fluorescent molecule absorbs a photon at a specific energy, it causes the molecule to enter an excited state, this state is unstable, then the molecule will enter into a less excited state, releasing energy in the form of light and heat in the process. This is fluorescence

Question 2

FLUOROPHORES

Answer 2

→ absorb light energy at a specific wavelength and re-emit it at a longer wavelength
→ the absorbed light, energy transfer efficiency, and time before emission all depend on fluorophore structure and chemical environment
→ emission energies range from ultraviolet to the visible emission spectrum, the spectrum can also continue into the infrared region
→ typically contains multiple aromatic groups

Question 3

USE OF FLUOROPHORES

Answer 3

→ they can be tracked
→ expression quantified
→ function determined
→ interactions studied

However they can be toxic!

Question 4

FLUORESCENCE MICROSCOPY (HOW IT WORKS)

Answer 4

→ specimen is illuminated with light of specific wavelength
→ light is absorbed by fluorophores and re-emitted as a longer wavelength
→ illuminated light is separated from weaker emitted fluorescence through emission filter

Question 5

DICHROIC FILTER

Answer 5

→ accurately filters colours, lets some colours pass and reflects away other colours

Question 6

LIGHT SOURCE

Answer 6

→ must be near monochromatic and intense. Ex. lasers, high power LED, xenon arc lamps, etc.

Question 7

NEGATIVE STAINING

Answer 7

→ sometimes specimens do not respond to electron beams, so negative staining is used to expose the negative space in the specimen. This is often done with biological molecules/ specimens.

Question 8

CRYOMICROSCOPY

Answer 8

→ Specimens are frozen to protect against radiation, however it must be done in a specific way so ice crystals don’t form, this can ruin the sample.