Magnetic nano-scale imaging of ferritin in a single cell

In life sciences, the ability to measure the distribution of biomolecules within an in situ cell is an important goal of investigation. Among a variety of techniques, scientists have used magnetic-based (MI) imaging based on the nitrogen vacuum (NV) center in diamonds as a powerful tool in biomolecular research. However, nanoscale imaging of intracellular proteins remains a challenge up to now. In a recent study now published in Progress of science, Wang Pengfei and colleagues from interdisciplinary physics departments, biomacromolecules, quantum information and life sciences in China, used ferritin proteins to demonstrate the realization by MI of endogenous proteins in a single cell, using the vacuum center Nitrogen (NV) as a sensor. They imaged intracellular ferritins and organites containing ferritin with the help of an MI and a correlative electron microscopy to pave the way for magnetic imaging at the nanoscale (MI) of intracellular proteins.

The increase in the existing spatial resolution of biomedical imaging is necessary to meet the current requirements of medical imaging. Therefore, magnetic imaging is of great interest for many techniques. Magnetic resonance imaging (MRI) is widely used to quantify the distribution of nuclear spins, but conventional MRI can only achieve a 1 μm resolution in nuclear spin imaging where the resolution is limited by the sensitivity of electrical detection. Scientists have developed a series of techniques to overcome this resolution barrier, including a superconducting quantum interference device and magnetic resonance force microscopy. Nevertheless, these reports require a cryogenic environment and a high vacuum for imaging, which limits the experimental implementation and its translation into clinical practice.

A newly developed quantum detection method based on the nitrogen vacuum center in diamond has radically pushed the boundaries of MI techniques to the nanoscale to detect organic molecules and proteins in the laboratory. Scientists have combined quantum detection with NV centers and scanning probe microscopy to demonstrate MRI at the nanoscale of a one – electron spin and a small nuclear spin. , while using the NV center as a biocompatible magnetometer for noninvasively imaging ferromagnetic particles in cells at the subcellular scale (0.4 μm). For example, depolarization of the center of the NV can be used as a broadband magnetometer to detect and measure the noise fluctuations of metal ions and nuclear spins. However, such individual protein imaging via a nano-scale MI has not yet been reported in the single cell.

In this work, Wang et al. reported two technical advances that allow nanoscale infiltration of intracellular proteins into a single cell. For this, they froze the cell on a solid state and segmented it into a cube shape, and then placed it on a scanning tuning fork of an atomic force microscope (AFM) for imaging purposes, where the flat cross section of the cell has been exposed to the air. Scientists used the sample positioning setup to allow the NV sensor to be positioned within 10 nm of the target proteins and the AFM to suppress thermal drift when positioning samples. They then developed trapezoidal cylindrical nanopillaries on a diamond surface in bulk for image acquisition, technically shortening the time of acquisition of images of an order by compared to previous methods. In the present study, scientists used this technique to perform in situ IM of the fluctuating magnetic noise of intracellular ferritin proteins (a biomarker of iron stores and transferrin saturation in the body) in the experimental setting.

Ferritin is a globular protein complex with an outer diameter of 12 nm, containing a cavity of 8 nm in diameter to store up to 4500 iron atoms in the protein. The magnetic noise of ferric ions can be detected because of their effects on the T₁ relaxation time of an NV center. In this work, Wang et al. confirmed the observation by using fluorescence measurements of the decay as a function of time of the NV center population (magnetic spin,_S = State 0), on a diamond surface covered with ferritines. In addition, scientists detected magnetic noise with unlabeled methods using the NV center using transmission electron microscopy (TEM). The work resulted in a correlated MI and TEM scheme for obtaining and verifying the first nanoscale MI of an in situ protein.

Scientists used the hepatic carcinoma cell line (HepG2) for experiments and studied iron metabolism by treating cells with ammonium citrate and iron (FAC), which significantly increased the amount of ferritin intracellularly. They verified this using first confocal microscopy (CFM), Western Blot and TEM techniques. The results showed the primary localization of ferritins in intracellular punctures around the nucleus, in the cytoplasm. Scientists used mass electron paramagnetic resonance spectroscopy (EPR) to confirm the paramagnetic properties of ferritin in FAC-treated HepG2 cells and mass spectroscopy to measure interference from other ions. paramagnetic metals.

Wang et al. We then used high-speed ultra-fast freezing to immobilize all intracellular components of Fe-loaded cells. The process stabilized intracellular structures and molecules by minimizing Brownian motion in the cells, which usually contributes to random movement proteins up to 100 nm in vivo. To image the samples, they incorporated and polymerized the frozen cells in LR White medium and then stuck the cell sample embedded on the AFM tuning fork with some cells at the tip. With the aid of a diamond knife, the scientists then cut the surface of the tip to a nanometer flatness to examine the section of cuboid cells under AFM. They acquired IM images of ferritins by scanning the cube of cells along the diamond nanopillars and simultaneously measured the spin repolarization rate of NV using the "jump" scanning mode of the microscope, as detailed previously.

Scientists measured the decay of fluorescence at a free evolution time of 50 microseconds (τ = 50 μs) to reveal the degree of spin polarization of the NV sensor, correlated with the amount of ferritin contained in the volume. detection. They observed the appearance of certain groups via the TEM and MI images, although some details were not observed in MI, the results confirmed that the spin noise of intracellular ferritin had contributed to depolarize the center of the NV. In order to obtain details on the ferritin clusters at a higher resolution, the scientists minimized the pixel size to 8.3 nm and acquired a high resolution MI of the proteins as expected.

In this way, Wang et al. explored the sensitivity of NV centers as the appropriate sensor for single-molecule biological imaging applications. They used the technique as a sensor in the experimental setup to obtain the first MI of a protein at a resolution of 10 nm in situ. Scientists aim to improve the stability and sensitivity of the technique in order to speed up the scanning process and to image a broader area of ​​interest in the cell and locate the ferritin beyond the nucleus in association with other organelles.

The work will contribute to clinical diagnoses aimed at determining the storage and release of iron based on biomarkers. This will include studies on the regulatory mechanisms of iron metabolism during the progression of hemochromatosis, anemia, cirrhosis of the liver and Alzheimer's disease. Wang et al. proposes to extend the in situ approach to other paramagnetic signal cell components, including magnetic molecules, metalloproteins and spin-labeled special proteins. Scientists plan to continue their studies by exploring other targets suitable for magnetic resonance imaging and magnetic resonance imaging techniques, as well as an optical microscopy detection integrated with the experimental setup, in order to Extend the work and determine the MRI of nuclear protein spin as well as three-dimensional cell tomography. .

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