Methods of probe microscopy used for magnetic resonance tomography and spectroscopy
immobilized on a hard substrate surface.
Nowadays, the crucial task in the spectroscopy of biological objects in the natural conditions and magnetic resonance tomography is to increase the spatial and time resolution and sensitivity. It may be solved by combining the methods of scanning probe microscopy and NMR spectroscopy. It was shown that the original hardware and software platform permits to measure simultaneously the morphology, structure and molecular composition of biomacromolecules immobilized on a hard substrate surface.
Scanning probe microscopy allows to visualize objects with atomic resolution in air and liquid [1,2]. Nowadays, methods of combined optical and scanning probe microscopy are actively used to increase the sensitivity, spatial and temporal resolution of magnetic and resonance imaging (MRI). Registration of spin magnetic resonance from a single protein molecular was carried out using nitrogen-vacancy center . Obtained is the image of a single atom of Ti with sub-micron spatial resolution  and different isotopes of Fe (56 and 57 a.m.u)  were registered using scanning tunnel microscope with the magnetic resonance. Besides, a specific arrangement of titanium isotopes placed on the MgO crystal adatoms surface was determined by magnetic resonance method. Atoms of nitrogen placed in vacancies of a diamond lattice may be used as the atom scale magnetometers and permit to measure polarization of nuclei in ensembles  and register the separate spins  including measurements in air under normal conditions .
The main idea of our project is to obtain the new data about structure and molecular composition of biological molecules (proteins, viruses, bacteria, cells) and their complexes in natural environments by applying MRI measuring equipment using the scanning probe microscopy. The key aim of our research is to develop and use special magnetic cantilevers with optimized precision signal registration methods to record signals in resonance and pulse modes in order to achieve the subnanometer spatial resolution in natural environments for biological objects such as air and liquids.
The unique design of cantilevers is due to use of:
- diamond-based probes with embedded atoms of nitrogen;
- probes with nano-scale ferromagnetic inclusions;
- probes with molecular composition of a tip based on the single atoms and their clusters.
Multifunctional scanning probe microscopy is applied to achieve a high spatial resolution in MRI due to a number of modes, such as scanning tunnel, scanning resistive, capillary, atomic power magnetic-force, Kelvin microscopy and electric power microscopy combined with the magnetic resonance tomography. To achieve a high temporal resolution, the high-speed electronics of the FemtoScan scanning probe microscope (see Fig.1) and the FemtoScan Online software  will be finalized and upgraded. In particular, a system for extracting a weak signal out of background noise using the SR844 synchronous amplifier (Stanford Research Systems, Great Britain) will be integrated into electronics. Measurements will be carried out using precision digital-to-analog and analog-to-digital converters of 20 bits capacity and a clock frequency of 1 MHz.
Use of the hyperpolarized Xe129 isotope to visualize cell structures, nanopores and ion channels in the cell membrane makes it possible to significantly increase the useful signal level. Xenon interacts with many different receptors and ion channels of cells and is a promising candidate as an anesthetic. Xenon is an antagonist of the NDMA receptor with high affinity to glycine site . Moreover, xenon is not neurotoxic and has a neuroprotective effect. It is planned to visualize inhibition of plasma membrane Ca2+ -ATPase by xenon and to study the spatial arrangement of other ion channels of the cell wall. For these purposes, an aqueous saline solution enriched by xenon Xe129 isotope will be applied.
Hyperpolarized xenon can also be applied to determine chemical composition of the surface. It is usually difficult to characterize surfaces using NMR because the signals from the surfaces are much weaker than the signals from atomic nuclei in the sample volume as far as their quantity is much greater than the surface nuclei number. However, it is possible to selectively polarize the nuclei spins on the solid surfaces by transferring on them the spin polarization from hyperpolarized xenon gas. This operation makes the surface signals sufficiently strong to measure them and distinguish from volume signals. Corresponding experiments may be carried out by using combined methods of scanning probe microscopy and nuclear magnetic resonance.
For local delivery of the solutions enriched by hyperpolarized xenon it is advisable to apply the capillary microscopy. We planned to use the diamond substrates with embedded atoms of nitrogen as quantum magnetometers. To analyze biomacromolecules, viruses and cells, the combined methods of atomic power microscopy and NMR spectroscopy should be applied. Additionally it is necessary to assess a possibility to use diamond probes with embedded atoms of nitrogen as a magnetic sensor (quantum magnetometer) with optical detection of the signal.
As a result, an experimental installation that combines possibilities of scanning probe microscopy and the spin resonance method will be developed to enable the following measurements and studies:
- measurements in controlled gaseous atmosphere of cell structures and model samples;
- measurements in helium atmosphere (xenon isotope 219) of cell structures and model samples;
- study of NMR contrast of cell membrane signal from the content of hyperpolarized xenon in the solution.
The study was carried out with the financial support of the Russian Foundation for Basic Research in the framework of scientific project No. 17-52-560001.