Fluorescense Flashcards
What can be done with fluor spectroscopy
Measure concentrations lower than absorbance can : nM and pM range
Do activity assays
Identify fluorophore
Measure exposure of fluorophores
Measure dynamics of fluorophores
Measure distances between fluorophores
Spectroscopic titrations: binding studies, like ITC and SPR
What is absorbance
How much light is absorbed by a substance
Also known as optical density (OD)
What is extinction coefficient (e)
What is the equation
How strongly light is absorbed by a substance at a given wavelength
Can be Represented as a molar extinction coefficient (molar concentration) or mass or other parameters
So at diff wavelengths that substance has diff e (absorbs less or more)
A=ecl
What is excitation
What is emmision
The ability of a molecule to absorb photons (light of a specific wavelength)
Release of photons from a molecule after excitation (usually released at lower energy)
What is quantum yeild
Probability that a fluorophore will emit a photon after excitation
Basically how likely it is to fluroense
When we excite a fluorophore where do we excite it
At the maximum absorbance peak (absorbance wavelngth max)
What is stokes shift
What is special about it
The difference between the excitation and emmision wavelength of a molecule
Wavelength em- wavelength ex
Stoke shift for a specific fluorophore can change based on the environment it’s in (polar vs non polar solvent)
What colour represents longer vs shorter wavelengths
What is the range of visible light
Red longer
Blue shorter
200-700
Explain the relationship between energy of emmision /excitation and wavelength of emmision /excitation
Explain the equation that says this
Energy of excitation is greater than energy of emmision
So
wavelength of excitation is lower than wavelength of emmision
E= hc/wavelngth
Energy decreases, wavelngth increases
Whag is the most prominent amino acid fluorophor we see in protiens
Max Wavelngth excitation and emmision
Quantum yeild
Trp
280 350
0.2 (large) means it emits a lot of light
What is special about adenine as a fluorophore
What is special about etbr
Has high extinction coefficient but very small quantum yeild
This means that although it absorbs a lot of light it doesn’t emit very much of the light
Etbr:
Has very high quantum yeild of 1 when intercalated between bases (very high emmision to detect dna)
Explain why a Fluor would have a larger stokes shift
The conformation of the protien
Solvent exposure:
more polar solvent (H2O), excited state of fluor loses energy to the polar solvent, lower energy of emmision means higher wavelngth of emmision, red shift (shift to longer wavelength)
Opp for less polar solvent: blue shift
What is the design of a fluorimeter
Light source: xenon or mercury arc lamps,
can also use laser of LED but less effective because don’t have broad nm excitation spectrum range
Monochromator: filter for specific wavelngth of both excitation light to hit sample and emmision light to hit deterctor
Emmision light at lower energy, more red, so can set the emmision monochroamtor to scan larger wavelngth ranges
Detector: has PMT to enhance the emmision signals that are transferred electronically into a spectrum
Describes the plot of trp excitation and emmision spectra
In a excitation / em does tea the fist peak is the excitation of trp at 280
Next peak is emmision at 350 (that is the trp)
A third peak showing at double the excitation wavelngth (560nm) is due to the excitation wavelngth undergoing a second order diffraction grating
How are the samples for fluroense prepared
2-3ml in a transparent cuvette (avoids interference from the cuvette in analysis)
Want the Absorbance at the excitation wavelngth < 0.1, so protiens concentration also need to be <0.1mg/ml
Want the Absorbance at the emmision wavelngth < 0.1
What are the types of cuvettes used in fluor
Traditional: 1 cm pathlength
Small volume: 0.2 cm pathlength
Why do we Want the Absorbance at the excitation wavelngth < 0.1
How do we avoid this
How do we avoid noise from tyrosine
If Aex wavelngth > 1, sample absorbs near the edge of the spectrum (not directly at the max excitation wavelngth), so emmision intensity decreases
Dilute samples , use smaller cuvettes,
Since greater absorabnce, more tyr noise, To avoid noise from tyr (since similar excitation wavelngth as trp) excite near the edge (at 295 nm) so that the tyrosine residue noise is avoided
Why do we want the Absorbance at the emmision wavelngth < 0.1
Due to the inner filter effect: after being emitted, light is reabsorbed by the sample (usually because of turbid sample) before reaching the detector
intensity of light going to the detector is decreased
Describe the inner filter effect
The detector usually collects light coming from the centre of the cuvette. In the inner filter effect the intensity of emmited light going to the detector is decreased
Two contributers to the inner filter effect:
Excitation light does not reach centre of cuvette
Emmision light does not reach the detector (is reabsorbed)
So at high abs values, there is a more rapid decrease in emmited light intensity
How do we fix inner filter effect
If can’t deal with the absorbance directly, use shorter pathlength cuvettes (so intensity doesn’t decrease as more as light goes through cuvette)
Dilute the turbid sample
What are the blanks and controls in fluor
A buffer blank to subtract signal from sample signal (for trp Fluor most buffers have very little fluor)
If looking for protien concentration you can use trp or NAWA as standards in a standard curve then measure your protien of interest to find its concentration
If your protien doesn’t have trp, you can use tyrosine in the standard curve
In measuring the emmision spectra of a protien, how is the excitation wavelength set
Can set the execution wavelength at the corresponding absorbance peak of your protien (example for trp set it at 280 cause that’s the peak)
If the fluorophore have a higher quantum yeild and extinction coefficient:
you can exited at the blue or red edge (shorter or longer wavlgnth)
This work too for If you only want to excite trp and not tyr residues, set the excitation wavelength to 295 nm (the red edge)
In measuring the emmision spectra of a protien, how is the excitation and emmision bandwaidth set
Usually between 1-10nm, Meaning if wavelength is 280 it’s 280 plus or minus 10 if bandwidth is 10
The IF (intensity of fluor) is proportional to bandpass squared:
So If 5nm to 1 nm, letting 10x less light into the cuvette which decreases signal
In measuring the emmision spectra of a protien, how is the emmision wavelength set
Between the wavelength of excitation and 2x wavelength of excitation
So that we don’t see ____
What do we do when the fluor intensity is too high?
Decrease excitation bandwidth (2x descrease = 4x less signal
Decrease concentration
Excite at the edge (change wavlgnth exciration)
Decrease PMT voltage (decrease sensitivity)
What do we do when the fluor intensity is too low?
Increase excitation bandwidth (excites more fluorophore)
Increase concentration
More limited in what you can do to increase the intensity
In measuring the excitation spectra, what do we do
What can this help with
Set the emmision wavlngth which is usually set at the peak emmision wavelength, and scan the excitation range
Can help characterize a fluorophore (but the excitation spectra is similar to the absorbance spectra)
can help understand energy transfer (how much energy is being lost if the fluorophore is in diff environments by the stokes shift of excitation to emmision spectra)
Explain the excitation spectra of NAWA
For trp: Fix the emmision wavelength (ex at 340 nm) then scan from 200-340 nm (the range of excitation)
So you need to have an idea of where the excitation happens to scan across that wavelength
Very star : big peak from the cuvette
280: see indole from NAWA
If see a small bump it’s Raman scattering
Bigger bump: Rayleigh scattering, this is a result of preferential scattering of materials at that certain wavelength
What are the steps to interpreting fluoresce spectra
Survey the x and y axis
Identify the peaks and fluorophore
Interpret red/blue shifts Kd the max wavlngth
Interpret intensity changes
Interpret energy transfer
Describe step 1 Survey the x and y axis
For the axis’s what is the wavelength / frequency (intensity) range
What are the units for the y axis fluorescence intensity (arbitrary, relative mostly used
Describe step 2 Identify the peaks and fluorophore
Use the max wavelength of em or ex to find the peaks
Beware of scattering
Describe step 3: Interpret red/blue shifts of the max wavlngth
If increase polarity, red shift, longer max wavelength , lower energy because water taking away energy from the excited state
Doesn’t always mean change in environment, can also mean more water making more polar environment
Describe step 4: Interpret intensity changes
If measuring quenching:
The flour intensity decreases if there is quenching from Cs, I, O2 or acrylamide)
Is the fluorophore avaible to the quencher?
Describe step 5 : Interpret energy transfer
If there is increased energy transfer, the Fluor intensity of the donor decreases and the acceptor increases
Fluorophores can transfer energy to other fluorophore, molecules, or solvent meaning protiens Tyr transfers energy to trp.
This makes direct measurement of tyr fluoresce hard unless trp is absent in the protien
Explain the apolipoprotien trp emmision shift before and after being mutated
They muted a trp into the helix and one into the loop
The spectra shows normal trp Fluor for native protien
If trp mutated into the alpha helix, shift to lower wavlngth (blue shift) and intensity decrease. This is because the trp in the helix is in a more hydrophobic environment , interacting less with water, has more energy of emmision , lower wavlngth
If trp mutated into loop region, see red shift. This is because the trp in the loop is more exposed to polar water (more polar environment), water takes energy from excited state, wavlngth increases
If the max em wavlngth of one peak (ex trp in loop) is more similar to the native protien, you would say that the native has a trp closer to the loop region than in the alpha helix
On Fluor spectra do we look the the Fluor of protien as a whole of the trp in the protien
How does this help in studies of protien
The trp
This is why interaction studies can happen: ex trp in substrate binding pocket, measure it’s fluor, introduce substrate that could remove water from the active site, see blue shift of the trp
What is Fluor quenching
Give example
Process the reduces Fluor intensity
Two solutions: Quinine tonic with high nacl, and with low Nacl
High nacl: the cl quencher the quinine fluor so less intensity
Low nacl: see emmision
How do we mathematically describe the quenching Kd a fluorophore with increases quencher
Stern volmer plot
What is a stern volmer plot
I0/I vs concentration of quencher
I0: fluor intensity at zero quencher
I: max intensity with quencher concentration x
The results show two types of quenching: static and dynamic
What is static and dynamic quenching
Static: the quencher bind to the fluorophore prior to excitation (M-Q Formed before excitation) , increases deactivation of M*
upward curve on stern volmer plot, exponential trend of quenching
Dynamic:
Collision between M* (excitation first) and that deactivates M*
Linear trend
What are the common quenchers
Cs, I, O2 acrylamide
Different quenchers work better on diff protiens
What is the stern volmer equation
What does it tell us
I0/I = 1 + Kq tao0 [Q]
y= b + mx
Tao0: is the excited state lifetime and a specific quencher concentration
Kq: quenching constant, tell is being more or less quenched (directly proportional to slope, bigger slope , bigger Kq, more quench)
When is Kq most accurate and applicable
Dynamic quenching because linear so you can get better numerical slope value
What does a buried fluorophore look like in stern volmer
The exposed trp are quenched at the beginning which is why linear start, but the buried are less accessible so quenching goes down
So you get downward curvature with state to level off because no more quenching occurring
Buried, smaller slope, smaller Kq, less quenching
Explain the stern volmer plots for trp accessibility in the apolipoprotien with trp in loop and trp in helix
All WT helix and loop is linear
Trp in helix (core of protien): the slope is not changing (the final and initial y values are similar, slope is very small) meaning the protien is not being quenched
Trp in loop (solvent exposed): highest slope, high values y values means greater intensity with no quencher than with, meaning more quenched
Explain the stern volmer plots for trp accessibility in the apolipoprotien with trp in loop and trp in helix
But when complex with lipids
Apolipoprotien s usually complex with lipids
In all three type of apolipoprotien, very little quenching happening meaning the binding of lipid reduces accessibility of trp to the quencher
What is another application of quenching and what does it mean
Ion analysis: finding ion concentrations
Making a standard curve of intensity vs increasing cl known concentration with quinine
Can to see concentration of cl in a environmental sample by using the curve
Explain automated quenching based screwing
A way to screen for which quenchers are for a specific molecule
Automated screening where the instrument set up all different quencher to be loaded in to sample cuvette
Makes an intensity vs time to tell which quencher results in quenching
Anything >50% reduction in fluor intensity is a potential quencher is
Explain automated quenching based screening
A way to screen for which quenchers are for a specific molecule
Automated screening where the instrument set up randomly positioned quencher to be loaded in to sample cuvette
Makes an intensity vs time to tell which quencher results in quenching (if quenched signal decrease)
Anything >50% reduction in fluor intensity is a potential quencher for that sample
What is self quenching
For example calcien:
High calcien concentration (>70 mM) three calcien molecules aggregate together
This aggregation has a low quantum yield (probability of releasing a photon is lower, not that it isn’t fluorescing)
At low calcien concentration,dilute, less aggregation, more intensity
What can self quenching allow for
How does to work
Leakage assays
These measure membrane stability
A liposome has calcien inside it at the self quenching concentration (looks brown)
When liposome less stable/broken, calcien leaks out, gets dilutes, increases Fluor (looks green)
Explain the calcien release example
Bound cobalt to calcien to stop its fluor (so not self quenching but cobalt quenched the calcien in the liposome)
A pore forming agent called mellitin makes pores in the membrane so that the calcien-co complex is released
Cobalt dissociates and bind edta in the solvent instead, calcien fluorescence occurs
First zero fluorescence, Increase mellitin, fluorescence increases, then add detergent to fully solbilize liposome and get 100% calcien release
This is to get a quantitative value from 0-100% to know how much leakage there is
Explain Laurdan fluoresce and the concept
Laurdan is a fluorescent probe that is highly sensitive to solvent polarity
Usually used for studies of membrane properties
Excite at 340nm, this makes an intermolecular dipole between the alkylamino and carbonyl group of Laurdan
Depending on the amount of water near that Laurdan dipole, the max emmision changes
If there’s more water exposure, the energy from Laurdan is transferred to water to allow the water dipoles to reorient in the same direction as the Laurdan dipole bc energetically stable (solvent relaxation)
Lower energy, higher emmision wavelength than 460 nm, red shift
Explain Laurdan in DMPG liposomes
In liposomes, Laurdan will also spontaneously insert itself into the liposome membrane through its alkyl chain
Laurdan senses how tight (ordered phase) vs loosely packed (disordered) the lipids are in the liposome
The Laurdan senses water coming in the liposome when disordered, the energy decreases (dipole reorientation) and wavelength increases
Explain how Laurdan in DMPG liposomes changes the emmision spectra
As temperature increases, measure the Laurdan signal at 440 and 490 nm
As temp increases, 440 signal decreases and 490 increases (because red shifting)
From these intensities get generalized polarization:
GP= I 440nm - 490nm / 440nm + 490 nm
GP Between 1 and -1, high values is ordered environment , lower values more disordered environment
What happens when you plot GP vs temp
Low temp, more rigid, gel phase, higher GP bc Laurdan less water exposed
Inflection point is melting temp
Higher temp, liquid crystalline phase, less ordered, Laurdan more water exposed, lower GP bc
How do GP plots and DSC plots relate
They both give similar melting temp values
Explain the GP vs temp for the Laurdan liposome but with cadmium metal added
More concentration of cadmium added, shift to right
- Cadmium Makes membrane more Rigid, GP values increase (bc intensity at 440 higher than 490 compared to control)
- The transition/melting temp shift to the right (15 degrees) because cadmium stabilized the rigid gel phase
Cd Stabilizing the gel phase is not good because DMPG is supposed to be fluid at physiologalc temp (37 degrees)
Charge of DMPG DMPC
DMPG negative
DMPC neutral
What is the ripple phase of a lipid
Some localized areas of movement, between rigid and gel phase Stabilizing the
