Importantly, since this information can be obtained from NV measurements performed on a representative sampling of cells or tissues, this obviates the need for NV evaluation of the exact individual sample imaged with MRI, enhancing the versatility of this approach. Predicted and experimental MRI behavior in cells. A library of 11 cells mapped with vector magnetometry three representative cells shown in a mix with unlabeled cells, was used to randomly fill a cell FCC lattice with periodic boundary conditions and run a Monte Carlo simulation of spin-echo MRI to predict T 2 relaxation behavior.
Our simulation predicted a bulk MRI T 2 relaxation time of Mixing was done to obtain a sufficiently long T 2 for accurate measurement with our MRI system. When compared to an experimental MRI measurement of T 2 in macrophages prepared as in the NV experiment and pelleted in a mixture with unsupplemented cells, the Monte Carlo prediction was accurate to within 2. The T 2 relaxation time of the cell pellets could not have been predicted solely from the concentration of IONs in the sample, as previous simulations have suggested a major influence of packing geometry on contrast agent relaxivity 8 , 9 , To establish that this relationship also holds for our model system, we performed MRI measurements and Monte Carlo simulations with IONs distributed in the extracellular space Fig.
Per iron mass, we found that this diffuse extracellular arrangement produces approximately sixfold faster T 2 relaxation than do endocytosed particles Fig. Simulations of additional particle distributions examine the relative influence of particle clustering and confinement inside cells and endosomes Supplementary Fig. To extend this technique to diagnostic imaging, we performed NV magnetometry on liver specimens from a mouse model of hepatic iron overload. The spatial distribution of iron deposits in the liver and other tissues has been a topic of interest in clinical literature as an indicator of disease state, including efforts to discern it noninvasively using MRI 2.
To investigate the microscale nature of this contrast enhancement, we cryosectioned the livers of saline- and iron-injected mice and imaged the magnetic field profiles of these tissue sections on our NV magneto-microscope. The magnetic particle clusters were relatively sparse, resulting in a punctate distribution of magnetic dipoles within the liver tissue of the iron-overloaded mouse Fig. We confirmed that these magnetic fields resulted from IONs using fluorescent imaging, for which purpose the IONs were labeled with a fluorescent dye Fig.
These results suggest that NV magnetometry could be used to map subvoxel magnetic field patterns within histological specimens, increasing the diagnostic power of MRI by correlating magnetic field distributions to disease state. Magnetometry of histological samples. Fluorescence images were taken with autogain to reduce the necessary exposure time, resulting in the visibility of the autofluorescence of the tissue in the saline control. Magnetometry scans were taken with a fixed gain. This experiment was repeated a total of three times, with data from two additional experiments shown in Supplementary Fig.
Finally, we tested whether NV magnetometry could be used to follow the magnetic consequences of the dynamic redistribution of magnetic material in living mammalian cells.
Macrophages endocytosing IONs go through several stages of internalization, gradually reconfiguring diffuse particles into compacted lysosomal clusters Fig. This process could be relevant to interpreting MRI data from labeled macrophages and to the development of clustering-based magnetic nanoparticle contrast agents 24 , To image living cells, we adjusted our NV methodology to minimize optical and thermal energy deposition.
This allowed us to generate time-lapse images of magnetic fields coalescing inside macrophages after ION internalization Fig.
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This technique for magnetic imaging of a dynamic cellular process could aid the development of dynamic contrast agents for MRI. Dynamic magnetic microscopy in live mammalian cells. Three additional replicates are shown in Supplementary Fig. Two additional replicates of this experiment are shown in Supplementary Fig.
In summary, this work establishes the capability of subcellular NV diamond magnetometry to map microscale magnetic field patterns in mammalian cells and tissues and introduces computational methods to connect these patterns to MRI contrast.
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The ability to make this connection experimentally will facilitate the interpretation of noninvasive images through microscopic analysis of matching histological specimens, and aid the development of magnetic contrast agents for molecular imaging and cellular tracking. Alternative methods for magnetic measurement, such as scanning superconducting quantum interference device SQUID microscopy 26 , 27 and magnetic force microscopy 28 , 29 , are more difficult to apply to tissue-scale biological specimens due to the need to raster scan samples, the spatial offsets required for thermal insulation of SQUID magnetometers from biological materials, and the need to penetrate samples with probe tips for force microscopy.
MRI itself can also be used at higher resolution to examine ex vivo specimens, but does not typically approach the single-micron level 30 , Meanwhile, methods such as electron microscopy or iron staining, which can also reveal the in vitro locations of putative magnetic materials based on their density or atomic composition, contain no information about the magnetic properties of such materials and their resulting fields, limiting the utility of these methods to examining the distribution of known magnetic field sources.
Although the present study also used known particles to enable direct experimental validation of our methods, NV magnetometry can in principle be used to measure magnetic field profiles arising from unknown sources, such as biomineralized iron oxide. To enable such measurements, NV imaging could be performed with a variable, electromagnet-driven bias field to first map the locations of magnetic field sources at low field where vector magnetometry is possible , and then apply a ramping field along a single NV axis to assess the M versus H behavior of each field source.
Such in situ saturation curves would provide the information needed to model MRI relaxation in samples with unknown saturation behavior. Additional improvements in this technique may be needed to reconstruct the location and magnetization of more diffuse magnetic materials that are less easily detected as point dipoles. This sensitivity was more than sufficient to detect the nm IONs used in our proof-of-concept experiments. While these particles are within the size range used in MRI contrast agents 21 , 22 , 23 , future work should focus on improving the sensitivity of NV magnetometry and demonstrating detection of smaller sources.
Sensitivity could be improved by employing diamonds with thinner NV layers, which would allow detection of significantly smaller magnetic sources near the diamond surface and would reduce the point-spread function of NV-imaged magnetic fields, increasing the precision of source localization. Combined with improved methods for positioning tissue sections flatter on the diamond surface, this would allow the mapping of fields produced by smaller, endogenous magnetic inclusions and ultrasmall superparamagnetic nanoparticles.
The study of microscale sources of T 2 contrast could be complemented by methods to map the concentrations of T 1 contrast agents using alternating current AC NV magnetometry In particular, adapting this technique to measure the 3D distribution of T 1 agents inside of the cell using nanodiamonds 32 , 33 could enable Monte Carlo modeling of T 1 relaxation in contrast-labeled cells and tissues. In addition to mapping the distribution of contrast agents and the resultant magnetic fields, recent advances in NV magnetometry could allow for in situ imaging of water-bound proton relaxation, enabling a direct measurement of the effect of contrast agents on the relaxation of surrounding water molecules Besides contributing to the study of MRI contrast, the methods presented for mapping magnetic field sources in 3D from planar optical data will enable biological imaging applications directly using NV diamonds and magnetic labels.
Because the optical readout in this technique is confined to the diamond surface, this method can be used to study opaque tissues inaccessible to conventional microscopy. To this end, our demonstration that time-resolved wide-field NV magnetic imaging can be performed on living cells increases the utility of this technique for monitoring dynamic biological processes. The top-surface NV sensing layer is measured to be 3. Layer thickness and nitrogen concentration were determined by secondary ion mass spectroscopy.
The diamond was irradiated with a 4. This diamond was affixed to a silicon carbide wafer for enhanced heat dissipation , which was in turn affixed to a pair of triangular prisms to facilitate a total internal reflection excitation path.
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The diamond assembly was removable to allow live-cell culture on the diamond surface in a cell culture incubator. Light was collected from the top of the diamond through a water-immersion objective.
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When necessary, focal drift was adjusted for using a piezo-driven stage Thorlabs. Microwave radiation was applied through a single turn copper loop immediately surrounding the diamond. The microwave signal was generated by a Stanford Research Systems Inc. The NV setup was controlled by custom software written in LabView.
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RAW For live-cell imaging, the cells were cultured as above until trypsinization and spotting on the diamond. Their media was supplemented with 0. The bias magnetic field was aligned close to in-plane with the diamond surface while having sufficient out-of-plane field strength to resolve the resonance of each NV axis, and the full NV optically detected magnetic resonance ODMR spectrum was probed.
The out-of-plane component was necessary because a purely in-plane bias field did not provide each NV axis with a unique parallel B -field, causing absorption lines to overlap. The microwave resonance for each pixel in the image was set as the center of the middle hyperfine peak of the transition. Spectra were swept at 0. Projection field maps were combined to form 3 orthogonal field maps with B z oriented normal to the diamond sensing surface.
B x is defined as the projection of the applied field onto the diamond plane and B y is defined along the vector that completes the orthogonal set. While probing only one NV transition allowed us to reduce the light dose to the sample while maintaining good SNR, it also limited our information to a projection of the field along one axis.
This limitation precludes the source fitting performed on the fixed samples. Regions of interest were selected to include all relevant fields for a given cell. Cell viability was assessed by performing a Trypan Blue exclusion assay after NV measurements. We performed inductively coupled plasma mass spectrometry ICP-MS to independently confirm the intracellular iron concentration estimated by NV magnetometry.
Unsupplemented cells contained 0.
In-plane dipole coordinates were identified as local minima in the B x field map. A pixel was identified as a local minimum if and only if its B x field value was smaller than all of its immediate neighbors including diagonals in the spatially low-passed image. All parameters are free to fit other than the in-plane dipole coordinates, which are fixed by the local minimum of the B x field map. While the z offset between the dipole and the diamond and the magnetic moment of the dipole both affect the strength of the detected field, they have distinguishable effects on the resultant field pattern.
After the strongest minimum has been fitted, the fitted field from the fit dipole within the full field of view was subtracted from the magnetic field image, to facilitate the fitting of weaker dipoles. The fitted field was subtracted, and the fitting continued until the list of local minima had been exhausted. A global fit was then performed using the results from the neighborhood fits as starting parameters.