Introduction to the Hole Burning Spectroscopy of Photosynthetic Chlorophyll-Protein Complexes.

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It all starts when the photon gets absorbed by the molecule (or atom, or aggregate...). Absorption of a photon results in the transition from ground to excited electronic state. The absorption spectrum of individual molecule consists of a narrow zero-phonon line (vibrational state of the system remains unchanged) and broad phonon sideband. If absorbing molecules are a part of a perfect crystal, all of them have the same local environments and all of them absorb photons of exactly the same energy (upper half of the picture). Real life is more or less far from that ideal case. In fact, all molecules have somewhat different local environments and somewhat different absorption energies (frequencies, wavelengths). Corresponding spectra are "inhomogeneously broadened" (lower half of the picture). (Modified figure by J. Friedrich, Technical University of Munich)
One can selectively excite molecules absorbing at given frequency using narrow-band laser. The excitation may lead to either photochemical transformation of the molecule itself or to rearrangement of its environment. In both cases the absorption frequency of the molecule changes. Total number of the molecules with absorption in resonance with the laser decreases. This phenomenon is called "persistent spectral hole burning". "Persistent" means that the new arrangement of the environment (or configuration of the molecule itself) is preserved for a long time after the laser is off. The smoke in the picture to the left is for entertainment purposes only, no "real" holes are burned into the samples. (Picture by M. Seebacher.)
The width of the spectral hole depends on the lifetime of the excited state. Which events determine that lifetime?
  • Emission of a photon. (Fluorescence.) Typically occurs within several nanoseconds for chlorophylls and similar molecules.
  • Dephasing - change of phase of the wavefunction in the excited state, for example due to interaction with phonons (delocalized lattice vibrations) or localized vibrations. Probability of such event is zero at 0 K but rapidly increases with temperature. In glasses and photosynthetic complexes dephasing occurs much faster than fluorescence even at several K. Therefore, in order to utilize high resolution of spectral hole burning one has to perform experiments at as low temperature as possible.
  • Energy transfer to other molecules (or aggregates). Occurs at picosecond and even femtosecond timescale in photosynthetic complexes.
    • As illustrated by the figure to the right, in the photosynthetic complexes the light energy is collected by a large number of antenna pigments and eventually transfered to the reaction center - the part of the photosynthetic unit where the charge separation occurs, the first step in a chain of reactions which result in formation of carbohydrates and molecular oxygen. 
    Next picture is a real life example. The spectra belong to Photosystem I of cyanobacterium Synechocystis. The width of a narrow hole at 670 nm (at the laser frequency) is determined by the rate of the downward excitation energy transfer. All photosynthetic complexes have well-defined structure (see below). It means that in every individual complex the molecules or groups of molecules  in the similar position within the complex absorb approximately at the same frequency. But only approximately, due to inhomogeneous broadening. The precise absorption frequencies of different molecules in an individual complex are not correlated. That is the reason why satellite holes in the figure are much broader than resonant hole. The widths of satellite holes are determined not by the lifetimes of corresponding states, but by inhomogeneous broadening.
    Summarizing, spectral hole burning can be utilized to determine:
    • The peak frequencies and inhomogeneous widths for various states (originating either from individual molecules or groups of molecules with small distances between them) - from satellite hole spectra or holeburning action spectra (wavelength dependence of the hole depth for fixed burning dose).
    • The energy transfer rates for different states - from the widths of resonant holes burned at various frequencies.
    • Temperature and pressure dependences of inter-molecular interactions and energy transfer rates.
    • Temperature dependence of dephasing - from the temperature dependence of the width of the hole burned into lowest-energy state (where downward energy transfer is impossible).
    • Parameters of electron-phonon coupling - from the detailed shape of spectral holes, including zero-phonon line and phonon sideband.
    • Difference in permanent dipole moments in the ground and excited states - from the broadening of the holes in electric field. The magnitude of that broadening is positively correlated with the magnitude of electron-phonon coupling and rates of pressure-induced shifts of spectral holes. Thus, it is possible to distinguish between states localized on individual chlorophyll molecules or delocalized over several closely spaced molecules.
    • And much more...
    And, finally, just some nice images of photosynthetic complexes:
    Photosystem I of cyanobacteria. Gray: antenna chlorophylls. Thick green, dark blue and light blue chlorophylls comprise the reaction center. Orange - Fe4S4 clusters. Thin lines - polypeptides. (Picture by A. Crofts, University of Illinois.)
     
    LH 2 antenna complex of purple bacteria.  Orange: B800 ring (absorption maximum at ~800 nm; excited states localized on individual bacteriochlorophyll molecules). Red: B850 ring (absorption maximum at ~850 nm; excited states delocalized over many bacteriochlorophyll molecules). Green: carotenoids. Blue spirals: polypeptides.