L12 - Optogenetic and Dyes

Question 1

What is Optogenetics?

Answer 1

Non-invasive

Uses light to modulate molecular events in a targeted manner in living cells or organisms.

Question 2

What kind of calcium indicators are exited by UV light?

Answer 2

Low-affinity: Fura-FF, BTC, Fura-2, Fura-5, Indo-1

Intermediate-affinity: Fura-4F, Fura-5F, Fura-6F

Question 3

What excites High affinity and selectivity (BAPTA)?

Answer 3

Visible light under scanning laser confocal microscopy:

Calcium Green
Calcium Orange
Oregon Green

Question 4

Fluorescence

Answer 4

molecular absorption of a photon triggers the emission of another photon with a longer wavelength.
o Electron can absorb a photon of light – electron goes higher layer.
o Blue light goes up to excite then when it drops to lower light goes to green light returns to eye.

Question 5

Fura-2-

Answer 5

• Most commonly used
• Calcium binds to a pocket and molecules swings together – arms come closer together – changes connectics of election and causes emitting of wavelength (the light back to use) to change.

Question 6

Fura-2- Problem and solution

Answer 6

Polar, binds to Ca but cant cross membrane

Add AM ester (non polar) cleaved after entry

FURA-2-AM

Question 7

Why are dyes important?

Answer 7

o Look at ion fluxes in real-time
o Determine spatial distribution of ion flux over multiple neurons
o Problem: still lacks specificity for cell type etc.

Question 8

Genetically-encoded Ca2+ indicators e.g. GCaMP (used by jelly fish)

Answer 8

• Latest developments: can be targeted to individual tissues/cells/domains
• Can have temporal control

Question 9

• Channelrhodopsin

Answer 9

o Rhodopsin from green algae (λ480nm) ion channel
o When in light stop when not flagella move

Chlamdomas stimulated by blue light

Question 10

• Halorhodopsin

Answer 10

o Rhodopsin from halobacteria (λ590nm) chloride pump
o Pump salt out
Activated by yellow light

7 transmembrane domain