Projects / Programmes
Molecular imaging inside the cell
| Code |
Science |
Field |
Subfield |
| 1.02.00 |
Natural sciences and mathematics |
Physics |
|
| Code |
Science |
Field |
| B191 |
Biomedical sciences |
Plant biochemistry |
| Code |
Science |
Field |
| 1.03 |
Natural Sciences |
Physical sciences |
imaging mass spectroscopy, secondary molecular ions, TOF, molecular imaging, MeV-SIMS, swift ions, electron desorption
Researchers (11)
Organisations (2)
Abstract
Imaging Mass Spectrometry (IMS) provides insight into biochemical processes by imaging the molecular distributions in biological tissues, shedding light on the complexity of the physiological processes. Available IMS technologies have significant limitations for imaging of molecular distributions on the subcellular level, as the probing mechanisms these techniques apply to extract biomolecules from the sample either destroy the weak bonds of biomolecules or are not capable of extracting the biomolecules with nanoscale lateral precision. The proposed project Molecular Imaging Inside the Cell (MICE) will develop novel and unsurpassed IMS technology, combining physics, biology, medicine and engineering, that exploits cutting-edge experimental developments in tissue cryoprocessing, high-energy focused ion beam formation, and mass spectrometry to capture images of molecular distributions within the cell. Images of non-fragmented molecular species heavier than 2 kDa will be recorded with lateral resolution of better than 500 nm, surpassing most of nowadays available IMS technologies. For the molecular probing mechanism, we will apply secondary ion emission induced by MeV heavy (swift) ions from the surface of insulating materials, the process explored in an emerging IMS technology known under the acronym MeV-SIMS (Secondary Ion Mass Spectrometry with MeV ions). By means of the existing ion optics focusing system at Ljubljana tandem accelerator, we will form high energy focused ion beams with the energy in the range between 4 and 10 MeV in the so-called low current mode, where the ion beam with the flux of 5000 ions per second and diameter of 500 nanometers strike the slice of biological tissue, penetrates it and hits a continuous electron multiplier, which triggers the measurement of the Time-Of-Flight. Dense energy transfer from swift ions to the electrons induces a high electron temperature burst along its trajectory, forming a propagating phonon wave, which softly desorbs non-fragmented secondary molecular ions from the sample. A focused beam of swift ions will be swept over the tissue maintained in a frozen hydrated state, and the induced secondary ions extracted in a reflectron Time-Of-Flight mass spectrometer. The resulting IMS technology will visualize intracellular chemistry with unprecedented details, bringing the lateral resolution down to the dimension of subcellular structures, and opening new frontiers in the understanding of the cell as the basic building element of life.
Significance for science
To overcome the existing limitations in the state of the art IMS to resolve molecular processes at subcellular scale, the project will develop new technology able to probe the molecular distribution down to 500 nm resolution, and deploy it for the imaging of selected physiological processes inside the cell, including some most scientifically interesting biochemical processes in vivo. Incorporating the probing mechanism based on electron desorption by swift ion impact featuring the combination of sub-500 nm lateral resolution and the ability of efficient non-fragmented desorption of large biomolecular ions, the technology developed within the project will enable molecular imaging with unprecedented lateral resolution and chemical sensitivity inside the cell, as well as the ability to measure analytical grade high-resolution mass spectra from so far unseen small volumes of samples on the order of 10-10 microliters. MICE will create unsurpassed visualisation technology revealing the biochemistry of cells and will complement new techniques of intracellular microscopies with nanometre resolution, being the first to image intracellular biochemistry through IMS. Incorporating high-resolution mass spectrometry, it will support the identification of the observed biomolecules, and enable discoveries of new biomolecules engaged in physiological processes. The members of the project team will be engaged in a demanding challenge to develop world leading technology, allowing as yet unseen visualisations of molecular processes inside cells, including signal transmission along synapses, responses of cells to synthetic chemicals and drugs, and the mechanisms of gene expression.
Project consists of several difficult and critical tasks, where new solutions and ideas need to be implemented to overcome bottlenecks in the available research methodology and technology in Imaging Mass Spectrometry, reaching the sub-100 nanometer resolution.
Among the most critical are: A) the formation of swift ion nanobeam with MeV energy and sub-500 nanometre diameter with quadrupole optics, B) technology of suspension of the frozen hydrated tissue over the background-free conductive substrate and its handling/transfer from the preparatory tools into the analytical environment.
These tasks will be addressed with a working team of members with interdisciplinary expertise, by exchanging and implementing their ideas, as well as in the intense communication with the research community working on these problems, also within undergoing and future international projects.
A very exciting pay-back is foreseen: the so-far unseen visualization of molecular distributions inside the cells, confirming or rejecting the existing models of intra-cellular metabolism, and opening new research subfield of subcellular molecular microscopy.
Significance for the country
To overcome the existing limitations in the state of the art IMS to resolve molecular processes at subcellular scale, the project will develop new technology able to probe the molecular distribution down to 500 nm resolution, and deploy it for the imaging of selected physiological processes inside the cell, including some most scientifically interesting biochemical processes in vivo. Incorporating the probing mechanism based on electron desorption by swift ion impact featuring the combination of sub-500 nm lateral resolution and the ability of efficient non-fragmented desorption of large biomolecular ions, the technology developed within the project will enable molecular imaging with unprecedented lateral resolution and chemical sensitivity inside the cell, as well as the ability to measure analytical grade high-resolution mass spectra from so far unseen small volumes of samples on the order of 10-10 microliters. MICE will create unsurpassed visualisation technology revealing the biochemistry of cells and will complement new techniques of intracellular microscopies with nanometre resolution, being the first to image intracellular biochemistry through IMS. Incorporating high-resolution mass spectrometry, it will support the identification of the observed biomolecules, and enable discoveries of new biomolecules engaged in physiological processes. The members of the project team will be engaged in a demanding challenge to develop world leading technology, allowing as yet unseen visualisations of molecular processes inside cells, including signal transmission along synapses, responses of cells to synthetic chemicals and drugs, and the mechanisms of gene expression.
Project consists of several difficult and critical tasks, where new solutions and ideas need to be implemented to overcome bottlenecks in the available research methodology and technology in Imaging Mass Spectrometry, reaching the sub-100 nanometer resolution.
Among the most critical are: A) the formation of swift ion nanobeam with MeV energy and sub-500 nanometre diameter with quadrupole optics, B) technology of suspension of the frozen hydrated tissue over the background-free conductive substrate and its handling/transfer from the preparatory tools into the analytical environment.
These tasks will be addressed with a working team of members with interdisciplinary expertise, by exchanging and implementing their ideas, as well as in the intense communication with the research community working on these problems, also within undergoing and future international projects.
A very exciting pay-back is foreseen: the so-far unseen visualization of molecular distributions inside the cells, confirming or rejecting the existing models of intra-cellular metabolism, and opening new research subfield of subcellular molecular microscopy.
Most important scientific results
Interim report
Most important socioeconomically and culturally relevant results
