ELM13: Optogenetics and dyes Flashcards
How do fluorescent reporter dyes work?
Substance binds to something else
Reporter dye changes optical properties
What is fluorescence?
Substance absorbs photon of light and emits light at longer wavelength
How does fluorescence work in a substance?
- Electrons excited by absorbing light
- Undergoes lots of small energy losses
- Drops down to lower state and emits light at lower energy level with longer wavelength
What are some uses of reporter dyes?
Harmless unlike electrophysiology
Can record multiple cells and circuits at a time
What is Fura-2?
Calcium fluorescent dye excited by UV light Ion sensitive
What is the structure of Fura-2?
Negative charges at top attract positive calcium ions
What happens when calcium interacts with Fura-2?
Calcium binds and distorts dye structure
Changes the spectrum of light Fura-2 absorbs and how it emits light
What is the absorption spectrum of Fura-2?
Type of light it will absorb
380nm in UV range
What is the emission spectrum of Fura-2?
Type of light Fura-2 emits
510nm in visible range
What occurs to the absorption and emission spectrum of Fura-2 when calcium is added?
Emission peak still at 510nm with increased intensity
Absorption peak at 340 and increased intensity
What is the process of using Fura-2 with calcium?
- Excite sample at 340 then 380 to stimulate with and without calcium
- Measure emission at 510nm for both
- Take ratio emission from 340 excitation /380
- Result is the measure of calcium
What is meant by the polarity of indicator dyes?
Polar so can’t cross cell membrane and enter dye
How do we overcome the barrier of indicator dyes not being able to enter cell?
Esterify the dye to make it non-polar
Can no longer bind calcium but enters cell
When inside esterases remove esters on COO- groups so they can bind calcium
What are genetically encoded calcium indicators?
Development in being targeted to single cells and tissues
Temporal control
A fusion protein
What are the uses of genetically encoded calcium indicators?
Make transgenic animal that expresses the receptor
Can target to tissue/cell specific promotor
What is fluorescence resonance energy transfer?
1.Uses one donor fluorophore and one acceptor fluorophore
2. Excited and emit at different wavelengths
3. Put in close proximity and donor excited using appropriate wavelength
4. Donor transfers energy to acceptor so emits fluorescence at characteristic wavelength
What are light activated channels and pumps?
Single celled organisms have channels powered by light so they can respond to it
What is ChR2?
Non selective cation channel
Built in retinal molecule
Blue light opens channel and influx of cations
What is NpHR?
Light powered chloride pump
Light hits retinal and chloride enters cell
What would be the effect of ChR2 in a neuron?
Influx of cations and depolarisation to fire AP
What would be the effect of NpHR in a neuron?
Membrane hyperpolarised due to influx of chlorine ions
What are OptoXRs?
Works in the eye but not in the brain
Can use chimeric proteins to make it work
What are chimeric proteins made up of?
Transmembrane and extracellular domain of vertebrate rhodopsin
Intracellular domain of desired GPCR
What is the method of radioligand binding experiments?
- Make radioligand of subject
- Mix with tissue of interest
- Measure how much radioactivity bound to tissue
What are the applications of radioligand binding experiments?
Adding radioligand to intact tissue
How tightly radioligand binds
What are some advantages of radioligand binding experiments?
Simple
Scaled up easily
Cheap
What are some disadvantages of radioligand binding experiments?
Doesn’t tell if drug is inhibiting or activating receptor
Bad time resolution
Hazardous materials and waste
