In life sciences, the ability to measure the distribution of biomolecules inside a cell in situ is an important investigative goal. Among other techniques, scientists have used magnetic imaging (MI) based on the nitrogen vacancy center (NV) in diamonds as a powerful tool in biomolecular research. However, nanoscale imaging of intracellular proteins has remained a challenge thus far. In a recent study now published in Science Advances Pengfei Wang and colleagues at the interdisciplinary departments of physics, biomacromolecules, quantum information and life sciences in China, used ferritin proteins to demonstrate the MI realization of endogenous proteins in a single cell, using the nitrogen-vacancy (NV) center as the sensor. They imaged intracellular ferritins and ferritin-containing organelles using MI and correlative electron microscopy for the nanoscale magnetic imaging (MI) or intracellular proteins.
Increasing existing spatial resolution of biomedical imaging is required to achieve ongoing demands in medical imaging, and therefore, among a variety of techniques, magnetic imaging or broad interest at present. Magnetic resonance imaging (MRI) is widely used to quantify the distribution of nuclear spins but conventional MRI can only reach a resolution of 1 µm in nuclear spin imaging where the resolution is limited by electrical detection sensitivity. Scientists have developed a series of techniques to break this resolution barrier, including a superconducting quantum interference device and magnetic resonance force microscopy. Nevertheless, these reports require a cryogenic environment and high vacuum for imaging, limiting the experimental implementation and its translation to clinical practice.
A recently developed quantum sensing method based on the nitrogen vacancy center in diamond has radically pushed the boundary of MI techniques to the nanoscale to detect organic molecules and proteins in the lab. Scientists have combined quantum sensing with NV centers and scanning probe microscopy to demonstrate nanoscale MRI for single electron spin and small nuclear spin ensemble while using the NV center as a biocompatible magnetometer for noninvasively image ferromagnetic particles within cells at the subcellular scale (0.4 µm). For example, depolarization of the NV center can be used as a wideband magnetometer to detect and measure fluctuating noise from metal ions and nuclear spins. However, such imaging of single proteins via MI at the nanoscale has not been reported in the single cell thus far.
In the present work, Wang et al. reported two technical advancements to allow nanoscale MI or intracellular proteins within a single cell. For this, they free-fixed the cell to a solid state and intricately segmented to a cube shape, then placed it on a tuning fork scanning probe or an atomic force microscope (AFM) for imaging, where the flat cross section of the cell was exposed to air. The scientists used the sample placement setup to allow the NV sensor to be located within 10 nm of the target proteins and used the AFM to suppress thermal drift during sample positioning. They then engineered trapezoidal cylinder-shaped nanopillars at a bulk diamond surface for image acquisition, technically shortening the time of image acquisition by one order compared to previous methods. In the present study, the scientists used this technique to conduct in situ MI of the magnetic fluctuating noise of intracellular ferritin proteins (a biomarker of iron stores and transferrin saturation in the body) within the experimental setup.
Ferritin is a globular protein complex with an outer diameter of 12 nm, containing a 8 nm cavity tension that allows up to 4500 iron atoms to be stored within the protein. The magnetic noise of ferric ions can be detected by their effects on the relaxation [of time] of an NV center. In this work, Wang et al. confirmed the observation using fluorescence measurements of time-dependent decay of the population of NV centers (magnetic spin, m S = 0 state), in a diamond surface coated with ferritins. Additionally, the scientists detected the magnetic noise with label-free methods using the NV center via transmission electron microscopy (TEM). The work allowed the development of a correlated MI and TEM to obtain and verify the first nanoscale MI of a protein in situ.
The scientists used the hepatic carcinoma cell line (HepG2) for the experiments and studied 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 first. The results showed the primary localization of ferritins in the intracellular puncta around the nucleus, among the cytoplasm. The scientists used bulk electron paramagnetic resonance (EPR) spectroscopy to confirm the paramagnetic properties of ferritin in the FAC-treated HepG2 cells and mass spectroscopy to the interference due to other paramagnetic metal ions.