Periodic Reporting for period 2 - smMIET (Single-Molecule Metal-Induced Energy Transfer (smMIET))
Período documentado: 2022-07-01 hasta 2023-12-31
The core aim of the project is to develop the technology of Single-Molecule Metal-Induced Energy Transfer (smMIET) for resolving macromolecular structure and dynamics with sub-nanometre spatial and nanosecond temporal resolution. Metal-Induced Energy Transfer or MIET was first developed in our group in 2012 for mapping cellular membranes with nanometre axial resolution. It exploits the effect that a fluorescent molecule, when brought close to a metal surface, can transfer its excited state energy to surface plasmons in the metal, which leads to a strong distance-dependence of its fluorescence lifetime and intensity. This strong lifetime-distance dependence allows for converting a measured fluorescence lifetime into a distance from the metal surface. Combining this concept with single-molecule localization super-resolution microscopy and with fluorescence correlation spectroscopy shall enable us to resolve three-dimensional molecular structures with nanometre isotropic resolution, and structural dynamics on the nanometre length scale with nanosecond temporal resolution. Among its many applications, the project aims to develop and apply smMIET for resolving the global structure of macromolecular complexes and their dynamics, the conformational fluctuations of intrinsically disordered proteins, the dynamics of lipid membranes, or the transport of proteins across lipid bilayers. Thus, smMIET will become an important tool for structural biology and biomedicine, which will enable to resolve the structure and dynamics of single biomolecules and biomolecular complexes with unprecedented spatial detail and temporal resolution.
So far, we have published 10 publications in peer-reviewed journals. In particular, we have published the following results:
1. Ghosh, Arindam et al. 2021. Nature Protocols 16 (7): 3695–3715. https://doi.org/10.1038/s41596-021-00558-6.
This paper gives a detailed step-by-step protocol how to implement MIET and GIET measurements in any interested lab, and it is thus of fundamental importance foe the wide dissemination of the method and will help other labs to implement it for a wide array of biological and biomedical applications.
2. Koenderink, A. Femius et al. 2021. Nanophotonics 11(2). https://doi.org/10.1515/nanoph-2021-0551
This paper is a review of the importance of plasmonics and plasmonics-based methods for biophysical and biomedical research, putting MIET and GITE ino a larger context and explaining the capabilities and prospects of our methods for resolving structures in biological samples.
3. Oleksiievets, Nazar, et al. 2022. Nano Letters, July. https://doi.org/10.1021/acs.nanolett.2c01586.
This paper presents a comparison between two methods of fluorescence lifetime single-molecule localization microscopy that were first developed in our lab and which are the technical basis for MIET/GIET imaging. We compare our wide-field fluorescence lifetime camera LinCAM with our rapid-scanning fluorescence-lifetime conofcfoal microscopy in the context of single-molecule localization and fluorescence lifetime multiplexing microscopy.
4. Oleksiievets, Nazar et al. 2022. Communications Biology 5 (1): 1–8. https://doi.org/10.1038/s42003-021-02976-4.
This is the first paper that presents a successful implementation of fluoresccence-lifetime single molecule localization microscopy for multiplexing. In particular, we were able to simultaneously image three different cellular structures based on different lifetimes of the used labels with nanometer lateral resolution. This is an important step towards full three dimensional MIET-based superresolution microscopy.
5. Raja, Sufi O. et al. 2021. Nano Letters 21 (19): 8244–49. https://doi.org/10.1021/acs.nanolett.1c02672.
In this paper, we applied GIET for monitoring the reorganization of the inner and outer membrane of individual mitochondria when switching from their inactive to their active state. We observe a relative change of the mean distance between the to membranes of only 2 nanometers in living mitochondria, which could not be observed with any other method. This demonstrates the enormous power of GIET for biophysical and biomedical research.
6. Sakhapov, Damir et al. 2022. The Journal of Physical Chemistry Letters, May, 4823–30. https://doi.org/10.1021/acs.jpclett.2c00896.
This is an important technical paper which presents a new and precise method for measuring the photophysical rates of fluorescent molecules. This will be of great importance for the correct data evaluation of MIET data when measuring the conformational dynamics of biomolecules.
7. Stallinga, Sjoerd et al. 2022. Physical Review Research 4 (2): 023003. https://doi.org/10.1103/PhysRevResearch.4.023003.
This paper is a fundamental theoretical study of the maximum spatial resolution of optical microscopy and present an algorithm for the most optimum data deconvolution of microscopy data. It is thus of fundamental importance for any microscopy modality.
8. Thiele, Jan Christoph et al. 2021. Science Advances 8 (December): 14190–200. https://doi.org/10.1101/2021.12.20.473473.
This is the first realization of three-dimensional MIET based super-resolution microscopy. We demonstrate that we can resolve with a combination of MIET with 2D single molecule localization microscopy the cylindrical structure of individual microtubules. It is the most important result and milestone of our project so far.
9. Thiele, Jan Christoph et al. 2021. Frontiers in Bioinformatics 1: 56. https://doi.org/10.3389/fbinf.2021.740281.
This is a technical paper discussing optimal data evaluation for fluorescence lifetime single molecule localization microscopy, which forms the basis of MIET/GIET super-resolution microscopy.
10. Yudovich, Shimon et al. 2022. Biophysical Journal 0 (0). https://doi.org/10.1016/j.bpj.2022.05.037.
In this paper we demonstrate a combination of MIET with a novel method of electrophysiology measurements for studying membrane potential and membrane organization in supported lipid bilayers.
1. Ghosh, Arindam et al. 2021. Nature Protocols 16 (7): 3695–3715. https://doi.org/10.1038/s41596-021-00558-6.
This paper gives a detailed step-by-step protocol how to implement MIET and GIET measurements in any interested lab, and it is thus of fundamental importance foe the wide dissemination of the method and will help other labs to implement it for a wide array of biological and biomedical applications.
2. Koenderink, A. Femius et al. 2021. Nanophotonics 11(2). https://doi.org/10.1515/nanoph-2021-0551
This paper is a review of the importance of plasmonics and plasmonics-based methods for biophysical and biomedical research, putting MIET and GITE ino a larger context and explaining the capabilities and prospects of our methods for resolving structures in biological samples.
3. Oleksiievets, Nazar, et al. 2022. Nano Letters, July. https://doi.org/10.1021/acs.nanolett.2c01586.
This paper presents a comparison between two methods of fluorescence lifetime single-molecule localization microscopy that were first developed in our lab and which are the technical basis for MIET/GIET imaging. We compare our wide-field fluorescence lifetime camera LinCAM with our rapid-scanning fluorescence-lifetime conofcfoal microscopy in the context of single-molecule localization and fluorescence lifetime multiplexing microscopy.
4. Oleksiievets, Nazar et al. 2022. Communications Biology 5 (1): 1–8. https://doi.org/10.1038/s42003-021-02976-4.
This is the first paper that presents a successful implementation of fluoresccence-lifetime single molecule localization microscopy for multiplexing. In particular, we were able to simultaneously image three different cellular structures based on different lifetimes of the used labels with nanometer lateral resolution. This is an important step towards full three dimensional MIET-based superresolution microscopy.
5. Raja, Sufi O. et al. 2021. Nano Letters 21 (19): 8244–49. https://doi.org/10.1021/acs.nanolett.1c02672.
In this paper, we applied GIET for monitoring the reorganization of the inner and outer membrane of individual mitochondria when switching from their inactive to their active state. We observe a relative change of the mean distance between the to membranes of only 2 nanometers in living mitochondria, which could not be observed with any other method. This demonstrates the enormous power of GIET for biophysical and biomedical research.
6. Sakhapov, Damir et al. 2022. The Journal of Physical Chemistry Letters, May, 4823–30. https://doi.org/10.1021/acs.jpclett.2c00896.
This is an important technical paper which presents a new and precise method for measuring the photophysical rates of fluorescent molecules. This will be of great importance for the correct data evaluation of MIET data when measuring the conformational dynamics of biomolecules.
7. Stallinga, Sjoerd et al. 2022. Physical Review Research 4 (2): 023003. https://doi.org/10.1103/PhysRevResearch.4.023003.
This paper is a fundamental theoretical study of the maximum spatial resolution of optical microscopy and present an algorithm for the most optimum data deconvolution of microscopy data. It is thus of fundamental importance for any microscopy modality.
8. Thiele, Jan Christoph et al. 2021. Science Advances 8 (December): 14190–200. https://doi.org/10.1101/2021.12.20.473473.
This is the first realization of three-dimensional MIET based super-resolution microscopy. We demonstrate that we can resolve with a combination of MIET with 2D single molecule localization microscopy the cylindrical structure of individual microtubules. It is the most important result and milestone of our project so far.
9. Thiele, Jan Christoph et al. 2021. Frontiers in Bioinformatics 1: 56. https://doi.org/10.3389/fbinf.2021.740281.
This is a technical paper discussing optimal data evaluation for fluorescence lifetime single molecule localization microscopy, which forms the basis of MIET/GIET super-resolution microscopy.
10. Yudovich, Shimon et al. 2022. Biophysical Journal 0 (0). https://doi.org/10.1016/j.bpj.2022.05.037.
In this paper we demonstrate a combination of MIET with a novel method of electrophysiology measurements for studying membrane potential and membrane organization in supported lipid bilayers.
We have developed two methods for fluorescence-lifetime single molecule localization microscopy (FL-SMLM). One is based on a new fluorescence-lifetime imaging wide-field camera, and the second on a rapid-scanning confocal microscope. We have thus, for the first time, opened the fluorescence lifetime dimension for single-molecule localization super-resolution microscopy, and we demonstrated the importance of this method for multiplexing and for MIET/GIET three-dimensional super-resolution microscopy. One of the most important results so far was the application of MIET-FL-SMLM for resolving clathrin-coated pits and the cylindrical structure of individual microtubules in biological cells. This is a landmark achievement and was one of the central goals of our project. Using GIET imaging, we were able to see mebrane re-organization of the membranes of individual intact and living mitochondria with nanometer resolution. We developed a new platform for the simultaneous measurement of structural details and electrophysiological properties of individual lipid membranes by combining MIET with electric potential measurements in a specially designed microelectronic chip.