CURRENT RESEARCH PROJECT

    Starting with Master of Science thesis at the Warsaw University through Ph.D. studies in the Institute of Physics Polish Academy of Sciences and first postdoctoral appointment at the University of Cincinnati I have been involved in studying the optical properties of various semiconductor structures, mainly quantum wells and quantum dots. However, somewhere in the process, seeing the importance of interdisciplinary research, I decided to gain experience in another, rather different discipline. As a result of this sudden twist, I am currently an Alexander von Humboldt research fellow in the Biophysical Chemistry Group at the Department of Chemistry and Biochemistry, Ludwig-Maximilian University Munich.
    Briefly, the focus of my research is to study single pigment-chlorophyll-protein complexes (in particular peridinin-chlorophyll-a-protein, PCP) using single molecule spectroscopy. More extended description of the first results could be found here.

1. Introduction.
    Proteins are biological molecules composed of linear chains of amino acids. This chain structure, even for small proteins, results in a large number of spatial degrees of freedom and possible structural configurations (conformations). Since each configuration of the amino acids corresponds to specific energy of a protein, the resulting energy diagram of the protein features a complicated and multidimensional landscape.
    In order to succinctly describe dynamics of the protein folding, a hierarchical energy funnel model has been proposed [1]. In this model, the structure of a protein fluctuates within the family of conformations characterized by similar energy while the transfer between different families occurs via non-equilibrium thermally activated transitions [1]. Protein folding is then a continuous transition from one conformation state to another, presumably with progressively lower energy. The photodissociation experiments, where large ensembles of myoglobin have been investigated, have indeed supported the energy funnel model [2,3]. However, a detailed description of the protein energy landscape requires the removal of the inhomogeneous broadening always present in ensemble experiments [4].
    The primary objective of my project is to apply single molecule spectroscopy to probe structural changes of chromophore - protein complex via monitoring the optical transitions of a single chromophore. Since such complexes are usually quite unstable and feature relatively low fluorescence quantum yield, I intend to improve collection efficiency of single molecules by introducing solid immersion lens (SIL).

2. Low Temperature Single Molecule Spectroscopy.
    In the pioneering papers it has been shown that low temperature single molecule spectroscopy is a powerful tool for probing interactions between an individual chromophore and its local environment [5]. Indeed, the influence of the surrounding has been inferred through monitoring the behavior of the zero-phonon-line (ZPL) absorption energy of a single molecule, while scanning the wavelength of the narrow-band excitation laser. Any modification of the chromophore’s environment can be observed through changes in either the energy or the intensity of the ZPL absorption. However, an important drawback of this method is that only molecules characterized with a sharp and spectrally stable ZPL absorption can be studied, which excludes most fluorescing systems, including fluorescent proteins.

    In order to overcome this serious limitation, I use an approach, in which a molecule is excited into its vibronic band, while the fluorescence of a very narrow ZPL is continuously monitored [6]. It has been shown that in this case the excitation efficiency remains nearly unaffected by even relatively large spectral jumps of the ZPL emission of a single chromophore (Fig. 1). This is highly important, as this single molecule excitation and detection scheme should be adaptable to any system, in which a spectrally broad absorbing state undergoes rapid conversion into an emitting state with a narrow ZPL linewidth.

3. Peridinin – Chlorophyll-a  – Protein Complex.
    Peridinin-chlorophyll-a-protein (PCP) complex is a photosynthetic molecule present in dinoflagellate Amphidinium carterae [7]. It is composed of a barrel of hydrophobic protein, which shields two chlorophyll-a molecules, each dressed by four peridinins (Fig. 2).

    This molecule features a very broad absorption band in the blue-green spectral range and, at the same time, fluorescence line narrowing shows narrow ZPL emission of chlorophyll (Fig. 3). For my experiment, it is important that energy relaxation processes from excited states down to the fluorescent state are extremely fast (<3ps) [8]. The ultrafast energy transfer is responsible for the absorption bands of the PCP complex being substantially broader than the ZPL emission of the chlorophyll.
    Taking all these properties of PCP into consideration, this complex seems ideal for the vibronic excitation scheme of single molecules. It should be possible to monitor the ZPL fluorescence of the chlorophyll while exciting through the broad absorption band around 500 nm composed of different short-lived states.

4. Solid Immersion Lens.
    One of the limitations of single molecule spectroscopy at cryogenic temperatures concerns usually low numerical aperture (NA) of a microscope objective. Low NA, while decreasing collection efficiency leads also to a large excitation volume and results in a more intense background. I will use a hemispherical solid immersion lens (Fig. 4), which will be placed between the microscope objective and the sample [9,10]. In this way, similarly to the oil immersion microscopy, the NA can be increased by n, where n is the refractive index of the SIL (in my case, 1.83). Since the working distance of the objective is only 0.4 mm, the SIL of a diameter of 0.5 mm will be used.

An important advantage of using SIL for studying biomolecules is that essentially the issue of an air gap between the SIL and the sample, which could severly destroy the optical performace of the SIL in the case of semiconductor nanostructures, is absent. The solution will be directly applied on the SIL flat surface and immediately frozen.

References
[1] H. Frauenfelder, S. G. Sligar, and P. G. Wolynes, Science, 254, 1598 (1991).
[2] A. Ansari, J. Berendzen, S. F. Bowne, H. Frauenfelder, I. E. T. Iben, T. B. Sauke, E. Shyamsunder, and R. D. Young, Proceedings of the National Academy of Science, 82, 5000 (1985).
[3] K. Fritsch, J. Friedrich, F. Parak, and J. L. Skinner, Proceedings of the National Academy of Science, 93, 15141 (1996).
[4] C. Hofmann, T. J. Aartsma, H. Michel, and J. Köhler, Proceedings of the National Academy of Science, 100, 15534 (2003).
[5] A. Zumbusch, L. Fleury, R. Brown, J. Bernard, and M. Orrit, Physical Review Letters, 70, 3584 (1993).
[6] A. Kiraz, M. Ehrl, Ch. Bräuchle, A. Zumbusch, Journal of Chemical Physics, 118, 10821 (2003).
[7] E. Hofmann, P. Wrench, F. P. Sharples, R. G. Hiller, W. Welte, and K. Diedrichs, Science, 272, 1788 (1996).
[8] T. Polívka and V. Sundström, Chemical Reviews, 104, 2021 (2004) and references therein.
[9] S. M. Mansfield and G. S. Kino, Applied Physics Letters, 57, 2615 (1990).
[10] S. B. Ippolito, B.B Goldberg, and S. Unlu, Journal of Applied Physics 97, 053105 (2005).

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