I. Fluorescence

A. As discussed last time (UV/Vis Spectroscopy) Molecules and atoms have different electronic states which differ in the distribution of electrons among various Atomic Orbitals (for atoms) and Molecular Orbitals (for Molecules)

  1. Ground State: electrons fill orbitals beginning with the lowest energy orbital pairing spins (2 electrons per orbital)
  2. Excited Electronic States: when a molecule has absorbed a photon with enough energy to cause an electron to jump from a lower energy orbital to an unoccupied orbital of higher energy -- the energy of the photon must be exactly equal to the energy difference, DE, between the ground state and the first excited single state (electron spins are still paired)

Energy-level diagram of two chromophores: G and S1 indicate the ground and first excited states respectively (heavy lines). Thevibrational levels are the thin lines. A. This molecule is capable of fluorescing by the transition (solid arrow) indicated in the diagram. After excitation, there are vibrational losses (wavy arrow) to the lowest leel of the excited state and then emission from this state (dashed arrow). B. This molecule fails to fluoresce because the vibrational levels of G are higher than the lowest level of S1; hence, there can be a nonradiative transition (horizontal wavy arrows) from S1 to a vibrational level of G followed by nonradiative losses to the bottom of G (vertical wavy arrow)

B. What happens to a molecule in the Excited Singlet State (ESS)? ESS can return to the Ground State (GS) in several ways; all involve giving up excess energy somehow.

  1. Internal Conversion: each electronic state has many vibrational energy states (and rotational energy states) of much smaller energy difference (corresponds to energy of Infra-Red photons). If an ESS vibrational energy level is close to a GS vibrational energy level, the electron can relax to the GS via transitions between vibrational energy levels giving off the excess energy to other molecules as heat (vibrational) energy.
  2. Fluorescence: the system can relax to the GS by re-emitting a photon. However, although the initial transition may be to a higher ESS vibrational energy level, the molecule will quickly relax to the lowest ESS vibrational energy level before returning to the GS with re-emission of a photon.
    1. emitted photon will have less energy than the photon absorbed ==> emitted photon has a longer wavelength, l.
    2. the photon will be emitted very quickly, within nanoseconds or picoseconds after excitation
  3. Intersystem Crossing: the electron goes to the Excited Triplet State (ETS), normally a rare event.
    1. Triplet State--lower in energy than ESS because of interactions between electrons with the same spin orientation
    2. transfer from Triplet State to Ground State is difficult because the electron spin must reorient to pair with the GS electron in the same orbital ==> ETS is long-lived, seconds to minutes
    3. Phosphorescence--emission of a photon during transition from ETS to GS. This takes much longer than fluorescence (seconds - minutes vs. nanoseconds to picoseconds) and is the phenomenon responsible for glow-in-the-dark paints etc. Very rare in solution because O2 is present at 100µM --> 1 mM and readily reacts with triplet states relaxing them without emission of a photon.

E. Instrumentation - Fluorescence: similar to a spectrophotometer; some spectrophotometers have accessories which convert them to fluorimeters. Select energy of excitation photon with the first monochromator and measure fluorescence emission spectrum with the second monochromator (scan with 2nd monochromator).

F. Terms and Parameters

  1. Fluorescence Quantum Yield = Q =(no. of molecules fluorescing) / (no. of molecules absorbing)
    Q is very difficult to measure precisely because the photomultiplier (detector) gives different outputs for photons with different l (this is not a problem with absorption spectroscopy because one measures ratios of intensity) ==> have to calibrate the photomultiplier by measuring its response to known intensities of light of different l.
  2. Fluorescence Lifetime, tF: excite with a very brief flash of light (<10-9 sec from a pulsed laser) to set up a population of molecules in the excited state. Then measure fluorescence emission as a function of time
    This will be a first order Kinetic decay ==> depends only on concentration of excited state molecules times some rate constant, k = 1 /

    in terms of fluorescence intensity, I: I = exp (t /tF) and k = 1/ tF

  3. 3. Q = tF/tR: What is tR ? It is the time constant for emission in the absence of competing processes (non-radiative etc.)

H. Sensitivity: Much greater sensitivity than Absorption Spectroscopy. Why?

  1. Absorption is a subtractive process. One measures small differences in large numbers (intensity of incident light -intensity of transmitted light). This difference will be very small for very low Absorbance from low concentrations. Best sensitivity is ~0.0005 OD but most spectrophotometers are not nearly this good.
  2. Fluorescence: Measures any detectable light against an essentially 0 background. Detectors are capable of measuring single photons ==> fluorescence can be extremely sensitive!

J. Intrinsic Fluorescence of Proteins: Due exclusively to Trp, Tyr, and Phe residues (unless the protein contains a fluorescent prosthetic group)

Phe -- Q very small ==> not usually useful

Tyr -- frequently quenched, especially if ionized or if near -NH3+ or -COO- or Trp

Trp -- most useful, lmax of emission shifts to shorter l (higher energy) and intensity increases when the polarity of environment decreases ==> Intrinsic fluorescence give information on protein conformation

  1. Measure fluorescence of Tyr and Trp ==> what their environment is like with respect to polarity (e.g. is it buried in a hydrophobic pocket); what groups are near
  2. Add an extrinsic fluorescence quencher to the protein solution ==> how does these affect the fluorescence of various Trp and Tyr residues; gives information on their environments, how exposed they are etc.

K. Extrinsic Fluorescence -- Reporter Groups

  1. ANS -- fluorescence is quenched in aqueous solution and greatly enhanced when bound in a hydrophobic pocket of a protein
  2. Reagents which react to form covalent bonds
    1. Dansyl Chloride -- sulfonyl chloride group reacts primarily with amino groups
    2. 1,5-I-AEDANS -- a derivative of iodoacetamide ==> reacts with Cys -SH groups
  3. Ethidium--fluorescence is greatly enhanced when it intercalates double-stranded DNA
  4. Fluorescent Indicators sensitive to:
    1. pH -- used to measure pH at enzyme active sites of in intracellular compartments
    2. Oxidation/Reduction Potential--voltage differences across membranes
    3. Ion Concentrations -- quin or fura used to measure intracellular [Ca++]

L. Applications of Extrinsic Fluorescent Probes

  1. Distance Measurement -- Single-Singlet Energy Transfer (a.k.a. Fluorescence Resonance Energy Transfer)
    1. need two chromophores- Donor (D) and Acceptor (A)
      If the energy of fluorescence emission by D overlaps the energy of Absorption by A, this energy can be transferred directly by interaction of the chromophore transition dipoles (dipoles induced during transition between electronic energy states). D's fluorescence emission transition state dipole excites A's absorption transition state dipole.
    2. Efficiency of this energy transfer depends upon:
      • degree of overlap of the energy levels of D and A ==> Spectral Overlap Integral
      • Relative orientations of the two transition state dipoles. Efficiency is highest when they are parallel and falls to 0 if they are perpendicular. If either D or A are free to rotate ==> orientation factor can be calculated for random orientations
      • 1/r6 -- where r is the distance between D and A. The goal is to determine r which is measurable over distances up to ~80 if spectral overlap is good.
    3. Measurement -- normally requires preparation and measurement of two otherwise identical samples containing: (1) D alone and (2) D and A
    4. three ways to measure efficiency of transfer
      • excite D and measure emission by A
      • measure decrease in Q (quantum yield) by D when A is present. Q decreases because some of the energy (some fraction of D* molecules) is transfered directly to A
      • measure decrease in tF (excited state lifetime) of D when A is present. tF decreases because of relaxation by transfer of energy to A
  2. Molecular Rotational Motion -- Fluorescence Polarization Anisotropy
    1. Excite with Plane Polarized Light ==> Photoselection
    2. the probability that a molecule will absorb a photon is highest when the electric vector, E, is parallel to the Absorption Transition Dipole Moment (µ) and falls to 0 when perpendicular. the Probability of Absorption is proportional to cos2q where q is the angle between E and µ. This produces a poplulation of molecules in the excited state with a particular orientation
    3. Measure Fluorescence Emission with a polarization parallel, I||, and Perpendicular, I^ to E ==> calculate either the:

    4. If molecules don't rotate, P and A have maximum values, Po and Ao, determined by the angle between Absorption and Emisison Dipoles. If this angle = 0 (a common situation) ==> Po = 1/2 and Ao = 2/5
    5. If molecules rotate during fluorescence emission lifetime (10's or 100's of nanoseconds, ns), then A (or P) start at Ao (or Po ) when t=0 and decrease with t at a rate determined by the rate of rotation of the fluorophore == Rotational Correlation Time. The equation describing this decrease for Anisotropy is: A(t) = Ao exp(-3t/r).
      This should be familiar, it is the same as the integrated rate law for a first order reaction, i.e.

      Here k = 3/r==> 3/ r is a first order kinetic rate constant for molecular rotation.

    6. ln A(t) = -(3/r) t + ln Ao ==> the slope of a plot of ln A(t) vs. t will be -3/ r and r, the Rotational Diffusion Coefficient, can be easily calculated.

  3. Translational Diffusion of Membrane Proteins: FRAP =
    ===> Fluorescence Recovery After Photobleaching
    1. requires a membrane of ~several microns diameter with fluorescent labeled proteins
    2. focus a laser on a small spot and bleach (destroy) the fluorophore in that area
    3. watch the rate at which neighboring fluorescent molecules diffuse into the bleached spot ==> calculate the 2-dimensional diffusion coefficient for that protein.