Instrumental2: 8 Fluro

What is the formula for quantum yield?

# of Luminescence photons/ # of Absorbance Photons

What is stokes shift?

The energy difference between the lowest energy peak of absorbance and the highest energy of emission.

What is Ro?

The forster radius, which is the distance at which energy transfer is 50% efficient

What is the inner filter effect in fluorescence?

At high concentrations of absorbing molecules, including the fluorophore the excitation of light is absorbed more. The result is that the intensity of the excitation light is not constant throughout the solution. Only a small percentage of the excitation light reaches the fluorophore of interest resulting in low emission

Why is fluorescence emission maximum usually at a longer wavelength than its absorption maximum?

Because the excited singlet state usually has time to relax to its ground vibrational state, so it doesn't fluoresce with as much energy -> less energy=longer wavelength

What are applications of GFP?

Can attach it to genes in other animals to make them glow, so we can tell gene expression

What are advantages of bioluminescence resonance energy transfer? BRET

Chemical rxn causes excitation, no light source needed. Spectral overlap isn't a concern and neither is photobleaching.

What can cause fluorescence quenching?

Collision with quenching molecules, emission transfer to non-fluorescent molecules, formation of a non-fluorescent complex

What is intersystem crossing?

Crossing from one spin state to another, usually from the first excited singlet state to the lower energy first excited triplet state.

What is fluorescence polarization?

Emission is polarized if molecular rotation is slow relative to fluorescence lifetime.

What is an example of FRET used?

Emission of an receptor before and after ligand is bound

What is bioluminescence?

Emission of light by living organisms due to enzymes and other proteins present in organsms

What are molecular luminescence methods based on?

Emission of radiation by a molecule that has reached an excited state by absorption of radiation

What are the steps in the processes of a photoluminescent system?

Excitation and Deactivation

What is vibrational relaxation?

Excited molecule transfers excess vibrational energy to the solvent, and quickly relaxes to the ground vibrational level in the excited state

What method can detect polymerization and hybridization?

Fluorescence correlation spectroscopy (FCS)

What is the difference between phosphorescence and fluorescence?

Fluorescence involves transitions that do not involve a change in electron spin and are short lived. It occurs usually from lowest excited state. Phosphorescence involves a change in spin and are longer lived.

What is chemiluminescence?

Fluorescence that occurs as a result of a chemical reaction. When the ground state of one molecule crosses with the excited state of a re-arranged or fragmented molecule.

What is fluorescence correlation spectroscopy? (FCS)

Fluorescent molecules diffusing in and out of a small volume result in fluctuations of the fluoresence intensity measured from that small volume element. Chnges due to brownian motion

What can FCS determine?

Fluorescent species diffusion coefficients, fluorescent molecules concentration, kinetic information of binding and aggregation processes

Where is energy in FRET transferred from?

From the donor molecule to the acceptor molecule, and the acceptor emits energy as light

Which states are most easily excited to their first excited singlet state?

Ground singlet states

What can lower fluorescence?

Heavy atoms in solvent, high charged groups and increase of oxygen or paramagnetic species.

How can we increase fluorescence with aromaticity?

Increase aromaticity=reduce internal conversion=higher fluorescence

What are disadvantages of BRET?

Limited number of donor-acceptor pairs available, and emission spectra is broad.

How can we increase fluorescence with intersystem crossing?

Lower intersystem crossing=higher fluorescence

How can we increase fluorescence with temp?

Lower temp=lower external conversion=higher fluorescence

How can we increase fluorescence with pie bonds?

More pie to pie start absorption transistions

What are characteristics of a spectrofluorometer?

More sophisticated and expensive. Uses monochromators. Tunable over many wavelengths.

Do all molecules fluoresce?

No many don't. It is good for analyzing complex mixtures that have fluorescent species. But not many compounds can by analyzed by this method.

Can transition in fluorescence take place from higher vibrational state?

No only from lowest.

Is the lifetime long in fluorescence?

No very short because spin is allowed transition, singlet to singlet

Is energy of emission greater than energy of absorption?

No, less.

How can we increase fluorescence with solvents?

Nonpolar solvents decrease external conversion=increase fluorescence

Is external conversion radiative?

Nope. Non-radiative

Is vibrational relaxation a radiative process?

Nope; Non-radiative

What is external conversion?

Transition between electronic states involving transfer of energy to toher species; Also referred to as quenching.

What's a chromophore?

Triad of amino acids Ser, Tyr, Gly. What gives the molecule color.

Is the spin usually paired or unapired in a triplet?

Unpaired. Lowest triplet usually T1.

How is FRET used in bioanalysis?

Useful tool to quantify molecular dynamics, such as protein-protein interactions, protein-DNA interactions, and protein conformational changes

Do singlet or triplet states last longer?

Usually triplet states stay in excited state longer

What is phorsphorescence?

When molecules in their excited triplet state can lose the energy back down to ground state without dropping to lower excited state first

Did they alter GFP to cover the full spectrum of colors?

Yes

Is fluorescent more sensitive than absorption methods?

Yes

Do fluorescent molecules have different lifetime?

Yes, a fluorescent molecule has different lifetimes and can be left to die out before the species of interest is measured

Can we use lasers in fluoro?

Yes, but need to be careful about sample decomposition such as photobleaching

Is FRET distance dependent?

Yes, the interaction between electronic excited states is

Is phosphorescence maximum usually longer than fluorescence?

Yes; Very long lived because its spin forbidden too

What is GFP?

an extremely stable and highly fluorescent protein used in research

What can bioluminescent proteins be used for?

markers or reporters in research and understanding physiological processes

What is quantum yield a measure of?

measure of efficiency of a luminescent system.

What are the three above requirements also related to?

The efficiency of FRET

What is a common accuracy w/ fluorescence?

2-10%

Which wavelengths can our eyes detect?

400-700

What brings about excitation?

Absorption of different bands of radiation

What is the most fundamental principle of FRET?

Absorption spectrum of acceptor must overlap fluorescent spectrum of the donor

What are three requirements of fluorescence resonance energy transfer (FRET)?

Acceptor and donor must be in close proximity; Absorption spectrum of acceptor must overlap fluorescence spectrum of donor; Donor and acceptor transition dipole orientations must be parallel

What is the principle behind fluorescence recovery after photobleaching? (FREP)

All fluorescent dyes emit light of one particular wavelength after they have absorbed light of another. However, if a high intensity blue light is shined to the dye, it will photobleach, meaning that the high intensity light renders it unable to fluoresce. This can lead to FRAP, which measures the ability of a molecule to move around over time

What differ between the phosphorimeter from spectrofluorometers?

Alternately irradiates sample and after a delay, measures phosphorescent intensity; Dewar surrounding sample cell cools it w/ liquid nitrogen reducing collisional deactivatioin

Is the spin usually paired or unpaired in a singlet?

Paired. Ground state usually a singlet.

What is P in the concentration dependence formula?

Power after traversing a length of medium

What is the F in the concentration dependence formula?

Power of fluorscence emission

What is Po in the concentration dependence formula?

Power of the incident beam soln

What is the greek symbol in the concentration dependence formula?

Quatum efficiency

What is internal conversion?

Radiationless decay. Molecules in an excited state can go to a lower state of the same energy. Loss of heat.

What are characteristics of filter fluorometer?

Simple, but limited use. Uses filters. Only one fixed wavelength of detection

What does a quantum yield of 1 mean?

That one absorbed photon is converted to luminescence, fuorescence or phosphorescence photons for every photon absorbed

What does excited state lifetime mean?

The amount of time it stays in the excited state

What is the quencher?

The chemical species which lowers quantum yield of luminescent process

What is there a delay between irradiation and measurement?

The delay allows the short lived fluorescence to pass through, so that only the longer lived phosphorescence is measured.