Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MeaSuRe (Following Protein Diffusion in Photosynthetic Membranes with Super Resolution)

Teaser

The regulated diffusion of light-harvesting complexes in photosynthetic membranes is essential for photosynthesis. To date, however, little is understood about the mobility of these proteins through the highly crowded membrane environment. The light-harvesting complexes...

Summary

The regulated diffusion of light-harvesting complexes in photosynthetic membranes is essential for photosynthesis. To date, however, little is understood about the mobility of these proteins through the highly crowded membrane environment. The light-harvesting complexes coordinate chlorophylls and are as such auto-fluorescent which makes fluorescence correlation spectroscopy (FCS) an attractive method to study their mobility. In this method the fluctuations in the fluorescence intensity when complexes move in and out of the diffraction-limited excitation spot is used to quantify the diffusion rates. However in the case of photosynthetic membranes there are two big challenges: 1) The concentration of the light-harvesting complexes in the membrane is extremely high, such that fluctuations are too small to observe. 2) The membrane dimensions are in the order of hundreds of nanometres which is the same as the diffraction limit of light, as such complexes will not diffuse in and out of the excitation spot. The aim of this project is to overcome these problems with the use of plasmonic nanoantennas which are able to confine light at the nanoscale. As a result only a small number of light-harvesting complexes will be excited, which will allow to measure fluctuations, and the excitation spot is smaller than the dimension of the membrane. This research is not only important for the understanding of photosynthesis, but the technique could also be applied to study cell membranes in general.

Diffraction limited FCS was used to measure the diffusion rate of light-harvesting complexes. In the second step the plasmonic nanoantennas were used to reduce the excitation volume. Plasmonic nano-gap antennas inside a nanoaperture were used to have strongly localized excitation and little background illumination. As expected, the excitation volume was reduced. However, the reduction was only a factor ten while a reduction of 4000 has been observed for organic chromophores. A possible problem is the size of the light-harvesting complex of about ten nanometre which might hinder the access to the antenna hot-spot. Different gap sizes of the antenna were investigated, but the results did not improve. Therefore, instead of gap-antennas rod-shaped antennas were used. With these antennas the maximal photon emission rate of single light-harvesting complexes was enhanced by a factor of fifty and the number of photons emitted before photobleaching was enhanced by a factor of ten. These antenna are promising candidates to use in the study of light-harvesting complex diffusion in a crowded membrane. Due to a new job after 9 month the project was stopped, therefore the fellow could not finish the entire project.

Work performed

Research results - At the start of the project the fellow received an advanced training in FCS measurements. Diffraction limited FCS was used to measure the diffusion rate of light-harvesting complexes. In the second step the plasmonic nanoantennas were used to reduce the excitation volume. As shown in the figure plasmonic nano-gap antennas inside a nanoaperture was used to have strongly localized excitation and little background illumination. As expected, the excitation volume was reduced. However, the reduction was only a factor ten while a reduction of 4000 has been observed for organic chromophores. A possible problem is the size of the light-harvesting complex of about ten nanometre which might hinder the access to the antenna hot-spot. Different gap sizes of the antenna were investigated, but the results did not improve. Therefore, instead of gap-antennas rod-shaped antennas were used. With these antennas the maximal photon emission rate of single light-harvesting complexes was enhanced by a factor of fifty and the number of photons emitted before photobleaching was enhanced by a factor of ten.

Training – With this project the fellow increased her experience with the management of a multidisciplinary research project and learned a new spectroscopic technique. She followed courses provided by Wageningen University to improve her teaching skills (Brain based teaching) and student supervision skills (Supervising MSc students). During her fellowship she supervised 2 MSc students with their thesis and co-supervised two PhD students. The fellow presented her work at several conferences and was involved in writing papers and grants. She strengthened her international network by keeping contact with the Institute of Photonic Sciences in Barcelona and improved her network by starting new collaborations with EPFL, Switzerland and Louisiana State University, USA.

Project management – The supervisor and the fellow agreed on a Personal Career Development Plan at the start of the project. For optimal development the fellow got the possibility to: supervise MSc students, co-supervise PhD students, take courses about teaching at Wageningen University, be involved in grant writing, write papers and present research results at conferences.

Impact – The fellow communicated about her research to high school students who are interested in the study Molecular Life Science and their parents. Short lectures were given at the Open Day of Wageningen University. In addition she was invited to talk about her research by the Molecular Life Sciences students during a lunch lecture. The fellow gained extra expertise in fluorescence measurements, confocal microscopy and the use of plasmonic antenna for biological research. Being awarded this MSC fellowship helped the fellow to obtain a tenure-track position at Wageningen University and to start her own research group in photosynthesis research.

Papers
Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy. Iermak I, Vink J, Bader AN, Wientjes E, van Amerongen H.
Biochim Biophys Acta. 2016 Sep;1857(9):1473-8. DOI: 10.1016/j.bbabio.2016.05.005

Pushing the Photon Limit: Nanoantennas Increase Maximal Photon Stream and Total Photon Number.
Wientjes E, Renger J, Cogdell R, van Hulst NF.
J Phys Chem Lett. 2016 May 5;7(9):1604-9. DOI: 10.1021/acs.jpclett.6b00491

Multiple LHCII antennae can transfer energy efficiently to a single Photosystem I. Bos I, Bland KM, Tian L, Croce R., Frankel LK, van Amerongen H., Bricker TM., Wientjes E. Biochim Biophys Acta. 2017 Feb 22; 1858(5):371-378. doi: 10.1016/j.bbabio.2017.02.012. [Epub ahead of print]

Conferences and other talks
Dutch Biophysics 2015, 28-29 September 2015, Velthoven, The Netherlands.

CHAINS 2015, 1-2 December 2015, Velthoven, The Netherlands. Selected presentation: Protein mobility in photosynthetic membranes

Light-Harvesting Satellite meeting of the 1

Final results

Plasmonic nanoantenna are an emererging tool in biological research. This work shows a potential new application for nanoantennas to study the protein diffusion on the nanoscale in biological membranes.