Nanoscale magnetic images of ferritin in a single cell



Nanoscale magnetic images of ferritin in a single cell

LEFT – Experimental configuration. The experiment was carried out in a homemade configuration, which combined magnetic resonance microscopy (ODMR) optically detected with atomic force microscopy (AFM). DM: dichroic mirror. BP: bandpbad filter operating at 650-775 nm. APD: avalanche photodiode. CCD: coupled loading device. LED: 470 nm light-emitting diode. AL: achromatic lens. PH: pin hole at a size of 30 μm. BS: beam splitter. RIGHT – Images of nanoplas on diamonds. (A) Obtaining SEM images of diamond nanopiles manufactured just after ion reactive etching (RIE). The upper part of the nanopillar is covered by hydrogen silsesquioxane (HSQ) to protect the NV center. (B) A single nanopilar in the form of a trapezoidal cylinder to detect sections of cells attached to the tip of the AFM. Scale bars, 10 μm (A); 400 nm (B). Credit: Science Advances, doi: 10.1126 / sciadv.aau8038.

In life sciences, the ability to measure the distribution of biomolecules within a cell in situ is an important research objective. Among a variety of techniques, scientists have used magnetic images (IM) based on the center of nitrogen vacancies (NV) in diamonds as a powerful tool in biomolecular research. However, the nanoscale image of intracellular proteins has been a challenge so far. In a recent study now published in Scientific advances, Pengfei Wang and colleagues from the interdisciplinary departments of physics, biomacromolecules, quantum information and life sciences in China, used ferritin proteins to demonstrate the realization of endogenous proteins in a single cell, using the nitrogen vacuum center (NV). ) as a sensor. Imaging of intracellular ferritins and organelles containing ferritin were used using MI and correlative electron microscopy to pave the way for the magnetic image (MI) at the nanoscale of intracellular proteins.

An increase in the existing spatial resolution of biomedical images is required to achieve continuous demands on medical images, and therefore, among a variety of techniques, magnetic images are of great interest at present. Magnetic resonance imaging (MRI) is widely used to quantify the distribution of nuclear spins, but conventional MRI can only achieve a resolution of 1 μm in nuclear spin images where resolution is limited by the sensitivity of electrical detection. Scientists have developed a series of techniques to break this resolution barrier, including a superconducting quantum interference device and magnetic resonance force microscopy. However, these reports require a cryogenic environment and a high vacuum to obtain images, which limits the experimental implementation and its translation into clinical practice.

A newly developed quantum detection method based on the center of nitrogen vacancies in the diamond has radically pushed the limit of nanoscale IM techniques to detect organic molecules and proteins in the laboratory. Scientists have combined quantum detection with NV centers and scanning-probe microscopy to demonstrate nanoscale magnetic resonance for a single electron spin and a small set of nuclear spin while using the NV center as a biocompatible magnetometer to capture ferromagnetic images Non-invasive cells within the subcellular scale (0.4 μm). For example, depolarization of the NV center can be used as a broadband magnetometer to detect and measure the fluctuating noise of metal ions and nuclear spins. However, such an image of individual proteins through MI at the nanoscale has not been reported in individual cells so far.

Nanoscale magnetic images of ferritin in a single cell

Outline of the configuration and experimental principle. (A) Schematic view of the experimental configuration. The resin-embedded cell is attached to a tuning fork and scans over the diamond nanopilar that contains a shallow NV center. A copper wire is used to send the microwave pulse to the NV center. A green laser (532 nm) of the confocal microscope (CFM) is used to address, initialize and read the NV center. (B) Left: crystal lattice and energy level of the NV center. The NV center is a point defect consisting of a substitute nitrogen atom and an adjacent vacancy in the diamond. On the right: schematic view of a ferritin. The black arrows indicate the electronic spins of Fe3 +. (C) Experimental demonstration of the detection of spin noise with and without ferritin in the form of polarization decay for the same NV center. The box is the sequence of pulses for the detection and image of ferritin. A green laser of 5 μs is used to initialize the state of rotation to ms = 0, followed by a time of free evolution τ to accumulate the magnetic noise, and finally the state of rotation is read by detecting the intensity of the fluorescence. The sequence of pulses is repeated approximately 105 times to acquire a good signal-to-noise ratio (SNR). The relaxation time is adjusted to 0.1 and 3.3 ms by exponential decay for the case with and without ferritin, respectively, which indicates a rotational noise of 0.01 mT2. Credit: Science Advances, doi: 10.1126 / sciadv.aau8038.

In the present work, Wang et al. reported on two technical advances to allow the nanoscale MI of intracellular proteins within a single cell. For this, they froze the cell to a solid state and intricately segmented it into a cube shape, then placed it on a scanning probe of a tuning fork of an atomic force microscope (AFM) to obtain images, where the cross section flat of the cell He was exposed to air. The scientists used the sample placement configuration to allow the NV sensor to position at 10 nm of the target proteins and used the AFM to suppress thermal drift during sample placement. They then designed trapezoidal cylinder-shaped nanopiles on a bulk diamond surface for image acquisition, which technically shortened the acquisition time of the image in an order compared to previous methods. In the present study, scientists used this technique to perform an in situ IM of the fluctuating magnetic noise of intracellular ferritin proteins (a biomarker of iron stores and transferrin saturation in the body) within the experimental setting.

Ferritin is a globular protein complex with an external diameter of 12 nm, which contains a cavity that covers a diameter of 8 nm that allows up to 4500 iron atoms to be stored inside the protein. The magnetic noise of ferric ions can be detected due to its effects on the Tone Relaxation time of a NV center. In this work, Wang et al. confirmed the observation by fluorescence measurements of the time-dependent disintegration of the population of NV centers (magnetic rotation, mS = 0 state), on a diamond surface coated with ferritins. In addition, the scientists detected magnetic noise with unlabeled methods using the NV center through transmission electron microscopy (TEM). The work allowed the development of a correlated MI and TEM scheme to obtain and verify the first nanoscale MI of a protein in situ.

The scientists used the liver carcinoma cell line (HepG2) for the experiments and studied the iron metabolism by treating the cells with ferric ammonium citrate (FAC), which significantly increased the amount of intracellular ferritin. They verified this using confocal microscopy (CFM), Western blotting and TEM techniques at the beginning. The results showed the primary localization of ferritins at the intracellular point around the nucleus, between the cytoplasm. The scientists used paramagnetic resonance spectroscopy (EPR) of mbad electrons to confirm the paramagnetic properties of ferritin in HepG2 cells treated with FAC and mbad spectroscopy to measure interference due to other paramagnetic metal ions.

Nanoscale magnetic images of ferritin in a single cell

TOP – The preparation and characterization of samples of HepG2 cells rich in ferritin. (A) Schematic view of the treatment of cultured cells. After iron loading or no treatment, HepG2 cells were examined for fluorescence images and EPR spectra, respectively. For MI and TEM images, the cell samples were treated by freezing at high pressure, freeze replacement and sectioning. (B) Representative confocal microscopy (CFM) image of ferritin structures (green) in HepG2 cells loaded with iron. The ferritin proteins were immunostained by an anti-ferritin light chain antibody. The nuclei are indicated by 4 & # 39 ;, 6-diamidino-2-phenylindole (DAPI) in the blue channel. The inset shows magnified ferritin structures. The dashed yellow line describes the outline of a cell. Scale bar, 20 μm. (C) EPR control spectra and HepG2 cells loaded with iron at T = 300 K. LOWER – Adjustment of the distance between the NV center and the cell section. (A) Interference strips between the cell cube and the surface of the diamond. Scale bar, 20 μm. (B) The geometric relationship and the R gap between cell samples and diamond abutments for MI. The diameter of the upper surface of the nanopilar is 400 nm. Credit: Science Advances, doi: 10.1126 / sciadv.aau8038.

Wang et al. then ultrafast high-pressure freezing was used to immobilize all the intracellular components of the Fe-laden cells. The process stabilized intracellular structures and molecules by minimizing Brownian movement in the cells, which typically contributes to the random movement of proteins up to 100 nm in vivo. To obtain images of the samples, they embedded and polymerized the frozen cells in LR White medium, and then pasted the sample of cells embedded in the AFM tuning fork with a few cells at the tip. Using a diamond knife, the scientists then sectioned the surface of the tip in nanometer to examine the section of cuboid cells beneath the AFM. They acquired IM images of ferritins by scanning the cell cube along the diamond nanoshells and simultaneously measured the repolarization rate of NV rotation using the microscope "head jump" scan mode as explained above.

Nanoscale magnetic images of ferritin in a single cell

Correlative images of MI and TEM. (A) Schematic view of the section for correlated MI and TEM images. The last section and the remaining cube were transferred for TEM and MI scan images, respectively. The section resulted in some divided ferritin groups that could be visualized in both microscopes. A transparent blue strip of ~ 10 nm indicates the IM image depth, while in TEM, the image depth is ~ 100 nm. (B) Ferritin distribution of the last ultrafine section under TEM. Box: Enlarged figure of the piece in black strokes box. (C) Result of MI of the remaining cell cube. The pixel size is 43 nm. (D) The combined micrograph of MI and TEM shows ferritins in a membrane-bound organelle. The red arrows in (B) to (D) indicate the same ferritin group. Scale bars, 5 μm (B) and 1 μm [B (inset), C, and D]. Credit: Science Advances, doi: 10.1126 / sciadv.aau8038.

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

In this way, Wang et al. explored the sensitivity of NV centers as an appropriate sensor for biological imaging applications at the single molecule level. They used the technique as a sensor in the experimental setup to obtain the first IM of a protein at a resolution of 10 nm in situ. The goal of the scientists is to improve the stability and sensitivity of the technique to accelerate the scanning process and visualize a larger area of ​​interest in the cell and locate the ferritin beyond the nucleus in badociation with additional organelles.

Nanoscale magnetic images of ferritin in a single cell

(A) Ferritin group with NV sensor images with 80 × 24 pixels and a pixel size of 8.3 nm. Scale bar, 100 nm. (B) Trace the data from the scan line in (A) directed by the red arrow. The platform indicates the ferritin group. The red curve adjusted by a plateau function serves as a guide for the eye. (C) Expanded figure of the discontinuous golden box in (B). The abrupt transition indicated by the red arrow around x = 283 nm shows the scan from the blank area to the area with ferritins. Credit: Science Advances, doi: 10.1126 / sciadv.aau8038.

The work will contribute to clinical diagnoses to determine the storage and release of iron-based biomarkers in cells. This will include studies on the regulatory mechanisms of iron metabolism during the progression of hemochromatosis, anemia, liver cirrhosis and Alzheimer's disease. Wang et al. It is proposed to extend the in situ approach to other cellular components with paramagnetic signals, which include magnetic molecules, metalloproteins and special proteins marked by spin. Scientists predict that more studies will explore additional targets suitable for high-resolution MI and related TEM imaging techniques, with the detection of optical microscopy incorporated into the experimental setup to extend the work and determine nuclear protein MRI and perform CT scans. three-dimensional cells.


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More information:
Mamin H.J. et al. February 2013, Science. Pengfei Wang et al. Nanoscale magnetic images of ferritins in a single cell, Scientific advances (2019). DOI: 10.1126 / sciadv.aau8038

Denis Vasyukov et al. A superconducting quantum scanning interference device with single-electron spin sensitivity, The nanotechnology of nature. (2013). DOI: 10.1038 / nnano.2013.169

D. Rugar et al. Detection of a single spin by magnetic resonance microscopy. Nature (2004). DOI: 10.1038 / nature02658

H. J. Mamin et al. Nuclear magnetic resonance at the nanoscale with a nitrogen vacuum rotation sensor, Science (2013). DOI: 10.1126 / science.1231540

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