the experiment performs the fastest linewidth measurement that is available [125]. However, due to the spectrally broad laser pulse many molecules within the heterogeneous broadened absorption line will be excited at the same time and ensemble averaging still takes place [127].
3.1.3 Single-molecule spectroscopy
In single-molecule experiments [44–46, 124, 129, 130] the ensemble averaging is completely eliminated. An individual molecule or pigment-protein complex residing in one and only one conformation can be studied. SHB and PE methods can derive information of the homogeneous linewidths of molecules and the rates of spectral diffusion processes. But only using single-molecule spectroscopy (SMS) information on the number of absorption lines that a single-molecule features and their relative intensities can be obtained. Spectral diffusion processes can not only be monitored in time but also in frequency space because it is possible to determine the spectral jump of an absorption since one can be sure that the absorption appearing at a different wavelength still belongs to thesamemolecule.
However, information can not only be gained about the properties of the molecules but also about their environment by using them as local probes. From the static part of the frequency shifts, information about the conformation of (matrix) molecules around the probe molecule or about proteins in the binding pocket of a chromophore can be obtained whereas from the time dependent part details about the couplings and characteristic times of the motions in the surrounding of the probe can be gained [45]. Studying these dynamics, time averaging has to be taken into account in single-molecule spectroscopy as sufficient photons have to be collected for an appropriate signal-to-noise ratio. However at low temperatures most dynamical processes are slowed down considerably making the time averaging less an issue [12, 52, 131, 132].
3.2 This thesis: single molecule spectroscopy
The purpose of this thesis is to study the static and dynamic properties of pigment-protein and pigment-pigment interactions in photosynthetic light-harvesting complexes. The number of absorption lines of a single complex, their spectral position and their homogeneous linewidths give information about the electronic structure of the different pigment pools and the type of
coupling within as well as about spectral diffusion processes caused by changes in the local environment of the pigments, for instance, due to protein motion. However, even at room temperature the absorptions of single molecules are generally so broad that they would overlap due to homogeneous line broadening. This broadening can be reduced by studying the samples at low temperatures, reducing the dephasing processes [44, 95, 133].
Therefore low-temperature single-molecule spectroscopy was chosen as experimental technique since it is the only method free from ensemble averaging that can distinguish whether absorp-tions belong to the same or to different molecules. Furthermore, photobleaching processes are strongly reduced at low temperatures allowing to study the complexes for a much longer time than at room temperature. Single pigments in these pigment-protein complexes are excellent candidates for probing the temporal fluctuations in their local environment which are normally hidden in the ensemble spectrum. Although SMS is not free from time averaging, the prob-lem of temporal resolution is lessened by decreasing the temperatures. At low temperatures the dynamics are slowed down and the linewidths of the absorptions are narrowed.
The question may arise about the relevance of cryogenic single-molecule spectroscopy. But at low temperatures very valuable relevant information about the structure and the energy landscape of the molecules studied is still available whereas the dynamics is slowed down to observable time scales. Furthermore, insights into basic interactions and processes of the molecules studied can certainly provide guidance for the interpretation of more complicated room-temperature observations [45, 46].
3.2.1 Prerequisites for single molecule spectroscopy
To obtain as large a signal as possible from a single molecule a number of conditions should be fulfilled: a large absorption cross section, high photostability, short radiative lifetime to ensure a high excitation turnover, low transfer probability into dark states such as triplet states, an operation below the saturation of the molecular absorption and a high fluorescence quantum yield.
Looking at the absorption cross section as defined in Eq. 2.2 τ2, CFC and CDW should be as large as possible. At sufficiently low temperatures (T <2K) most phonon modes are not pop-ulated which means thatτ2∗ becomes extremely long and thereforeτ2 equals2τ1 [95] (see also
3.2: This thesis: single molecule spectroscopy 31 Eq. 2.4). In addition, electron phonon coupling is weak and the Debye-Waller factor becomes large [44, 105]. For the zero-phonon line of an aromatic molecule the linewidthΓhomdecreases from∼ 1000cm-1 at room temperature to∼ 10-3cm-1at cryogenic temperatures and is orders of magnitude smaller than that of a typical phonon side band (several 10 cm-1) [95, 133]. Taking into account the higher Debye-Waller factor as well, the peak absorption cross section reaches up to106A˚2atT < 2K, 5 orders of magnitude larger than at room temperature and correspond-ing to about 100 000 times the molecular size [95]. As the lifetime of vibronic levels of large molecules in a matrix is very short (picoseconds) their vibronic ZPLs are very broad compared to those of pure electronic ZPLs (lifetimes of nanoseconds). Therefore in low temperature sin-gle molecule excitation spectroscopy experiments only the pure electronic ZPLs show up and their narrow linewidths make detection against the background noise much easier [44, 95].
All together, the number of photons that are emitted from a chlorophyll molecule can be as high as 100 000–1 000 000 per second [24, 101, 102], see section 2.2.1. However, the great difficulty is to separate these photons from all the photons emitted by the other molecules around the chlorophyll. Optical bandpass filters can block most of these background photons at the cost of loosing also a significant number of the signal photons. This leads to a typical detection efficiency of a low-temperature single-molecule setup of well below 0.1% which only allows to detect at maximum a few hundred photons of a chlorophyll molecule per second.
3.2.2 Techniques in low temperature single molecule spectroscopy
A short history
In 1989 Moerner and Kador [34] were the first to optically detect a single molecule at low temperatures using a sensitive doubly modulated absorption technique. Soon afterwards, Orrit and Bernard [35] showed that fluorescence-excitation spectra enhanced the SNR dramatically compared to the absorption spectra. Here, a laser excites a small sample volume, in which at most one molecule is present that can absorb at the wavelength of the incident light and only the red-shifted fluorescence is collected from the same volume. This selective fluorescence detection allows to efficiently reduce the residual stray laser photons as well as spectral features due to impurities or Raman scattering of the matrix or the solvent and a signal is observed that arises from a single molecule.
To reduce the background even further, the excitation volume of the sample has to be made as small as possible and the collection efficiency of the fluorescence as large as possible. Various optical schemes have been developed for this purpose. For instance, in the first fluorescence-excitation experiment [35] the sample was glued to the end of an optical single mode fibre limiting the excitation volume to the core diameter of 2µm of the fibre. Other solutions com-prise a small lens [134] or parabolic mirror [135] to focus the laser beam onto the sample, or a pinhole [136] to limit the incident laser beam. The collection of the fluorescence has to be performed over a wide solid angle as it is emitted in all directions. Concave parabolic mirrors [88], objectives [137] or simple lenses [134] were used for this purpose. The excitation volume can be decreased even more by taking advantage of the geometry of the sample and using for instance thin films [132] or nanocrystals [138].
Confocal microscopy
Nowadays, the most commonly used experimental setup for single molecule spectroscopy is the confocal microscope. As depicted in Fig. 3.1, the excitation light is focused onto a pinhole which is subsequently imaged onto the sample by a high numerical aperture objective causing a small focal volume of about 1µm3 (1 femtolitre). The fluorescence is collected by the same objective, imaged onto a second pinhole — thereby blocking emission light from out of focus regions — and finally focused onto a detector. In this way, the confocal detection scheme ensures that only photons are detected that arise from the sample volume which is confocal with both the excitation as well as the emission pinhole, efficiently suppressing unwanted scattered light and fluorescence from areas outside the focal volume.
sample objective dichroid excitation pinhole
excitation volume
detection pinhole
detector
Figure 3.1:Confocal principle. As the sample volume is confocal with both excitation and detection pinhole, only the excitation volume is illuminated and light from out-of-focus regions is efficiently blocked.