Spectroscopic Properties of Photosystems Revealed by Single-molecule Spectroscopy at Low Temperature

In this thesis, single-molecule spectroscopy (SMS) at low temperature is utilized to comprehensively investigate the spectroscopic properties of the light-harvesting antenna complexes such as photosystem I (PSI) and II (PSII). The fluorescence of single PSII core complexes (PSIIcc) shows that the spectra are dominated by sharp lines, which come from multiple emitters and not only from one lowest trap. These datasets show the existence of the three well-known fluorescence bands denoted F685, F689, and F695. Further analysis of dimeric PSIIcc (dPSIIcc) presents that these sharp lines are the result of weak to intermediate exciton-vibrational coupling and slow spectral di usion. The Huang-Rhys factors, which are a measure of the strength of exciton-vibrational coupling, vary between 0.03 and 0.8 in single dPSII. Based on these values, there is no obvious correlation between coupling strength and wavelength position. These results show that electrostatic, rather than exchange or dispersive interactions, are the main contributors to the exciton-vibrational coupling in dPSII systems. The SMS of single monomeric PSII core complexes (mPSIIcc) at 1.6 K, also shows the same three fluorescence bands detected in dPSIIcc, however, the intensity of F695 in mPSIIcc SMS datasets is reduced as compared to the single dPSIIcc. The mPSIIcc contains one Beta-Carotene less than dPSIIcc at the monomer-monomer interface of dPSIIcc, which leads to an increased lifetime of the triplet state by Chl 17. This explains the reduced singlet emission of F695 in mPSIIcc. The results from fluorescence measurements of single PSIIcc and PSI complexes show different spectroscopic properties. The fluorescence of single PSI complexes are mostly characterized by sharp lines and a broad emission at 1.6 K. Polarization dependent SMS provides detailed insight into the fluorescence dynamics of the red Chls, demonstrating that Chls absorb light at longer wavelengths than reaction center, and their interactions with other chromophores are mainly happened by excitation energy transfer (EET). The spectral dynamics between the single PSI trimer (PSI-T) and monomer (PSI-M) complexes in both S- and P-polarization dependent datasets indicate that the single PSI-M complexes feature less spectral diversity of their fluorescence than single PSI-Ts. The analysis of polarization dependent datasets from single PSI-M complexes demonstrates two spectrally separate emissions corresponding to two fluorescence pools in PSI-M. Representatively, an almost perpendicular orientation is estimated between them, where they are not connected with each other via energy transfer pathways. Finally, suitable candidates for red-coupled Chl dimers based on the X-ray structure of PSI-M are discussed. The fluorescence enhancements of PSI-T in proximity to bimetallic plasmonic nanostructures are reported using SMS at cryogenic temperature (1.6 K). Moreover, controlled modifcation of fluorescence and energy transfer properties of PSI complexes are shown by using a Fabry-Perot resonator with silver mirrors. Finally, resolution enhancement of a confocal scanning microscope using immersion liquid under cryogenic conditions for imaging the biological specimens is discussed

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)

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 β-carotene (β-Car) cofactors located at the monomer–monomer interface in dPSIIcc is missing in mPSIIcc. This β-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 β-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

Effects of Irregular Bimetallic Nanostructures on the Optical Properties of Photosystem I from Thermosynechococcus elongatus

International audienceThe fluorescence of photosystem I (PSI) trimers in proximity to bimetallic plasmonic nanostructures have been explored by single-molecule spectroscopy (SMS) at cryogenic temperature (1.6 K). PSI serves as a model for biological multichromophore-coupled systems with high potential for biotechnological applications. Plasmonic nanostructures are fabricated by thermal annealing of thin metallic films. The fluorescence of PSI has been intensified due to the coupling with plasmonic nanostructures. Enhancement factors up to 22.9 and 5.1 are observed for individual PSI complexes coupled to Au/Au and Ag/Au samples, respectively. Additionally, a wavelength dependence of fluorescence enhancement is observed, which can be explained by the multichromophoric composition of PSI

Orientations between Red Antenna States of Photosystem I Monomers from <i>Thermosynechococcus elongatus</i> 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 <i>a</i> (Chl <i>a</i>) molecules responsible for the fluorescence emission of photosystem I monomers (PS I-M) from the cyanobacterium <i>Thermosynechococcus elongatus</i>. The fluorescence spectra of single PS I-M are dominated by several red-shifted chlorophyll <i>a</i> 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

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