Role of missing carotenoid in reducing the fluorescence of single monomeric photosystem II core complexes.

The fluorescence of monomeric photosystem II core complexes (mPSIIcc) of the cyanobacterium Thermosynechococcus elongatus, originating from redissolved crystals, is investigated by using single-molecule spectroscopy (SMS) at 1.6 K. The emission spectra of individual mPSIIcc are dominated by sharp zero-phonon lines, showing the existence of different emitters compatible with the F685, F689, and F695 bands reported formerly. The intensity of F695 is reduced in single mPSIIcc as compared to single PSIIcc-dimers (dPSIIcc). Crystal structures show that one of the ß-carotene (ß-Car) cofactors located at the monomer-monomer interface in dPSIIcc is missing in mPSIIcc. This ß-Car in dPSIIcc is in van der Waals distance to chlorophyll (Chl) 17 in the CP47 subunit. We suggest that this Chl contributes to the F695 emitter. A loss of ß-Car cofactors in mPSIIcc preparations will lead to an increased lifetime of the triplet state of Chl 17, which can explain the reduced singlet emission of F695 as observed in SMS.

Resolution enhancement for low-temperature scanning microscopy by cryo-immersion.

Here we report a simple way to enhance the resolution of a confocal scanning microscope under cryogenic conditions. Using a microscope objective (MO) with high numerical aperture (NA = 1.25) and 1-propanol as an immersion fluid with low freezing temperature we were able to reach an imaging resolution at 160 K comparable to ambient conditions. The MO and the sample were both placed inside the inner chamber of the cryostat to reduce distortions induced by temperature gradients. The image quality of our commercially available MO was further enhanced by scanning the sample (sample scanning) in contrast to beam scanning. The ease of the whole procedure marks an essential step towards the development of cryo high-resolution microscopy and correlative light and electron cryo microscopy (cryoCLEM).

Spectroscopic properties of photosystem II core complexes from Thermosynechococcus elongatus revealed by single-molecule experiments.

In this study we use a combination of absorption, fluorescence and low temperature single-molecule spectroscopy to elucidate the spectral properties, heterogeneities and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence emission of photosystem II core complexes (PS II cc) from the cyanobacterium Thermosynechococcus elongatus. At the ensemble level, the absorption and fluorescence spectra show a temperature dependence similar to plant PS II. We report emission spectra of single PS II cc for the first time; the spectra are dominated by zero-phonon lines (ZPLs) in the range between 680 and 705nm. The single-molecule experiments show unambiguously that different emitters and not only the lowest energy trap contribute to the low temperature emission spectrum. The average emission spectrum obtained from more than hundred single complexes shows three main contributions that are in good agreement with the reported bands F685, F689 and F695. The intensity of F695 is found to be lower than in conventional ensemble spectroscopy. The reason for the deviation might be due to the accumulation of triplet states on the red-most chlorophylls (e.g. Chl29 in CP47) or on carotenoids close to these long-wavelength traps by the high excitation power used in the single-molecule experiments. The red-most emitter will not contribute to the fluorescence spectrum as long as it is in the triplet state. In addition, quenching of fluorescence by the triplet state may lead to a decrease of long-wavelength emission.

Manipulating the excitation transfer in Photosystem I using a Fabry-Perot metal resonator with optical subwavelength dimensions.

We demonstrate controlled modification of the fluorescence and energy transfer properties of Photosystem I (PSI) - one of the most important light harvesting systems - by using a newly developed approach to produce optical subwavelength microcavities for cryogenic temperature issues. The experiments were carried out on PSI from the cyanobacterium Arthrospira platensis as it shows a broad and structured fluorescence emission. By changing the distance between the cavity forming mirrors, the electromagnetic field mode structure around PSI is varied affecting the emission and energy transfer properties, which allows us to selectively enhance signals of resonant emitters and suppress off-resonant emission. By comparing the experimental data with simulations, we are able to show how excitation transfer within PSI is affected by the microcavity. The ability to control the energy transfer within such efficient energy converters as photosynthetic proteins can establish the opportunity for enhancing the efficiencies of bio-solar applications. The defined control of the resonance conditions by microcavities makes them a preferable tool to study the effects of additional electromagnetic modes on the energy transfer in any coupled multi-chromophore system. The resonator geometry excludes the direct contact of the proteins with any surface. Possible quenching or denaturation of the complexes close to metal surfaces is still an insuperable obstacle for studies with proteins and nanostructures, which can be avoided by resonators.

Temperature dependence of metal-enhanced fluorescence of photosystem I from Thermosynechococcus elongatus.

We report the temperature dependence of metal-enhanced fluorescence (MEF) of individual photosystem I (PSI) complexes from Thermosynechococcus elongatus (T. elongatus) coupled to gold nanoparticles (AuNPs). A strong temperature dependence of shape and intensity of the emission spectra is observed when PSI is coupled to AuNPs. For each temperature, the enhancement factor (EF) is calculated by comparing the intensity of individual AuNP-coupled PSI to the mean intensity of 'uncoupled' PSI. At cryogenic temperature (1.6 K) the average EF was 4.3-fold. Upon increasing the temperature to 250 K the EF increases to 84-fold. Single complexes show even higher EFs up to 441.0-fold. At increasing temperatures the different spectral pools of PSI from T. elongatus become distinguishable. These pools are affected differently by the plasmonic interactions and show different enhancements. The remarkable increase of the EFs is explained by a rate model including the temperature dependence of the fluorescence yield of PSI and the spectral overlap between absorption and emission spectra of AuNPs and PSI, respectively.

Orientations between Red Antenna States of Photosystem I Monomers from Thermosynechococcus elongatus Revealed by Single-Molecule Spectroscopy.

Single-molecule spectroscopy at low temperature was used to study the spectral properties, heterogeneities, and spectral dynamics of the chlorophyll a (Chl a) molecules responsible for the fluorescence emission of photosystem I monomers (PS I-M) from the cyanobacterium Thermosynechococcus elongatus. The fluorescence spectra of single PS I-M are dominated by several red-shifted chlorophyll a molecules named C708 and C719. The emission spectra show broad spectral distributions and several zero-phonon lines (ZPLs). Compared with the spectra of the single PS I trimers, some contributions are missing due to the lower number of C719 Chl's in monomers. Polarization-dependent measurements show an almost perpendicular orientation between the emitters corresponding to C708 and C719. These contributions can be assigned to chlorophyll dimers B18B19, B31B32, and B32B33.

Variation of exciton-vibrational coupling in photosystem II core complexes from Thermosynechococcus elongatus as revealed by single-molecule spectroscopy.

The spectral properties and dynamics of the fluorescence emission of photosystem II core complexes are investigated by single-molecule spectroscopy at 1.6 K. The emission spectra are dominated by sharp zero-phonon lines (ZPLs). The sharp ZPLs are the result of weak to intermediate exciton-vibrational coupling and slow spectral diffusion. For several data sets, it is possible to surpass the effect of spectral diffusion by applying a shifting algorithm. The increased signal-to-noise ratio enables us to determine the exciton-vibrational coupling strength (Huang-Rhys factor) with high precision. The Huang-Rhys factors vary between 0.03 and 0.8. The values of the Huang-Rhys factors show no obvious correlation between coupling strength and wavelength position. From this result, we conclude that electrostatic rather than exchange or dispersive interactions are the main contributors to the exciton-vibrational coupling in this system.