DIAMOND MAGNETOMETRY
A review of the bioanalysis research group at the University Medical Center Groningen
Ruben van Harmelen
S3000702
Ruben.v.harmelen@gmail.com Bachelor thesis
Supervisor: dr. Romana Schirhagl Bioanalysis research group
Date: 05/07/2018
1
Abstract
Nanodiamonds have been shown to have very unique high potential properties. Apart from many industrial applications (e.g. abrasives, catalyst), they also found their way into the medical world.
Specifically, nanodiamond containing Nitrogen-Vacancy (NV) centers are very versatile promising new probes in correlative microscopy. These high-pressure high-temperature (HPHT) nanodiamonds in size tens of nanometers reveal to have an inert diamond structure, yet a highly functionalizable surface chemistry and a good internalization potential and biocompatibility. Their versatility in imaging lies in the possibility of cathodoluminescence and the virtually unlimited photo stability property which is also dependent on magnetic fluctuations up to a single electron or nucleus spin.
Thus, the path to visualize on the atomic scale may have been paved. The behavior of fluorescent nanodiamond in vitro has been examined and protocols to counter aggregation in medium have been proposed. When cells do not readily take up the nanoparticles, a chemical transformation protocol has been found, which cause the cells minimum distress. In-depth research in the diamond dimensions give us insight into a flake-like diamond structure, which helps us understand biological process like endosomal escape through their relative sharp shape, as well as physical processes, like behavior in magnetic field as effect of the aspect ratio of the particle. Nanodiamonds could prove to be superior probes, replacing the currently used inferior probes like quantum dots. Also, very new imaging techniques may arise, through the possibility of visualizing magnetic fields on the nanoscale. With all this said, just de surface is scratched of the possibilities of nanodiamonds, before nanodiamonds can be declared the golden standard of imaging, much more fundamental research should be done towards in-vivo cytotoxicity and creating more efficient nanodiamonds (smaller and brighter).
2
Table of content
Abstract ... 1
Introduction ... 3
Diamond magnetometry ... 3
Bioanalysis ... 5
Towards applied diamond magnetometry ... 6
Article overview ... 6
Nitrogen-Vacancy Centers in Diamond: Nanoscale Sensors for Physics and Biology10 ... 7
Nanodiamonds as multi-purpose labels for microscopy16 ... 8
The interaction of fluorescent nanodiamond probes with cellular media17 ... 9
Generally Applicable Transformation Protocols for Fluorescent Nanodiamond Internalization into Cells18 ... 10
Shape and crystallographic orientation of nanodiamonds for quantum sensing3 ... 11
Discussing and future perspectives ... 12
Acknowledgments ... 12
References ... 12
3
Introduction
Diamond magnetometry
Visualizing internal processes in cells is a complex thing to do. Light microscopy encounters barriers in diffraction limits, which leads to insufficient magnification and magnetic resonance imaging (MRI) encounters limitations in magnetic field sensitivity.1 In last two decades we found a high potential technique for biomedical imaging possibly surpassing above mentioned limitations. Diamond
magnetometry is a way to visualize magnetic fields using diamonds. These diamonds contain nitrogen- vacancy’s (NV)2, a nitrogen-vacancy is a nitrogen atom and an empty spot in the structure within a diamond particle (See Figure 1B for illustration). A NV-center can contain a free electron, which is revered to as NV- center, this NV- center is fluorescent when irradiated with a laser. This NV- is also magnetic.
For diamond magnetometry we use very small diamonds, called nanodiamonds (NDs). NDs range in size from a few nanometers to around a hundred Nanometer.3 The structure and surface of this particles can be modified, so that it interacts in a functional way (think about making the particle
‘stick’ to a certain surface). This is further revered to as functionalization.4 Keeping above mentioned in mind, these fluorescent nanodiamonds (FNDs) which are magnetic may play a crucial role in the context of biomedical imaging.
Currently organic dyes or quantum dots (semiconductor particles such as silicon) are used as probe in biomedical imaging. However, these dyes quickly lose their fluorescence, because the
fluorescence component in the dye is damaged by light, higher temperatures or fixation processes used in biological research (this is called photobleaching). Quantum dots are much more stable but their toxicity is quite unclear, but it tends towards being toxic.
The characteristic of the fluorescent nanodiamonds of having the NV-centers embedded in their lattice diamond structure2, gives the FND the property of fluorescence which is resistance to photobleaching and fixation processes.5,6 Because the NV-center is very small and magnetic, it allows for measuring very small magnetic changes on the nanoscale, which is a form of called quantum sensing. This can be done at room temperature. Also, FNDs have an excellent biocompatibility.6,7 Above mentioned features combined, potentially make FNDs a great probe or sensor for internal cell processes. FNDs can be used to sense many kind of processes, like thermal fluctuations, electric fields and most interesting magnetic fluctuations.8–11This last process is something the Bioanalysis groups mainly focusses on and is used for diamond magnetometry.
Diamond magnetometry can be used to visualize processes where free radicals are produced (e.g. energy production by mitochondria, aging or cellular stress), which causes damage in cells12, since they are very reactive.13 These radicals are very short living particles, usually hard or impractical to visualize.14 But with diamond magnetometry, magnetic field fluctuations up to 0.36 μT can be detected15, which is sensitive enough to detect the magnetic field of free radicals. To be able to do this, means a step closer of understanding many diseases on the cellular level (e.g. cancer), which on its turn possibly leads to the development of new, more effective medicine.
In diamond magnetometry a laser excites the FND, which then start to fluorescence. External magnetic field fluctuations influence the intensity of the fluorescence which is being recorded. This is called Optically Detected Magnetic Resonance (ODMR), so basically magnetic fields can be
visualized.16 The setup to record ODMR is in essence a confocal microscope combined with
microwave electronics to apply a magnetic field (Figure 1A).10 Figure 2 shows a schematic overview on how a NV-center is recognized. The excited and ground state are divided by three sub-spin levels.
When a magnetic field of 4.4mT is applied, a differentiation is seen in two of the sub-spin levels. This is characterized by the double dip in luminescence intensity in Figure 2C. The most useful fluorescent luminesce originates from negatively charged NV-centers (NV-), so usually NV- are discussed in this paper. Non-charged NV-centers (NV0) or positively-charged NV-centers (NV+), also contribute to fluorescence, but since these are not magnetic, they are useless for diamond magnetometry.10
4
Figure 2. Characteristics of the nitrogen-vacancy (NV) center. (a) Energy-level diagram of NV−. |g> denotes the electronic ground state, |e> the strong and weak nonradiative decay via the singlet state |s>. Wiggly arrows indicate the radiative transition. Electron paramagnetic resonance (EPR) spectrum of a single NV center at zero and nonzero magnetic field, recorded using the optically detected magnetic resonance technique (b, c).
By reference 10
Figure 1. (A) Schematic setup to readout ODMR, by reference 16. (B) Lattice structure of the nitrogen-vacancy, V = Vacancy, N = Nitrogen, C = Carbon, by Reference 10
5
Bioanalysis
Since the discovery of nanodiamond particles and their possible applications, a huge amount of research is done towards these particles. In this case particularly fluorescent nanodiamonds for diamond magnetometry. But because the interest in these particles is relatively new, only little is known about the detailed behavior of these particles in biomedical usage. The aim of the Bioanalysis research group is to study these particles and its behavior in multiple aspects e.g. how do fluorescent nanodiamonds behave in living cells, what is the surface characterization of the particles, how are the diamonds taken up, how do we control them and how to practically apply diamond magnetometry?
This review will consider different papers published by the research group and give a clear insight into what have been researched, why it was relevant for diamond magnetometry, what we learned from it and what is still lying in the future. A top-down structure is applied in writing this review: The first paper will lay down some basic knowledge about nanodiamonds, to better grasp the rest of this review’s content. Secondly, microscopy will be discussed which is a bio-physical section.
Followed by the interaction and properties of nanodiamonds, deepening a bit more the biological and (bio)chemical aspect and finally the fundamental properties of NDs will be reviewed going into the particle physics. An overview of the content division is summarized on the next page (Table 1.
Overview of a set of articles published by the bioanalysis research group, Figure 3. A systematic overview on different aspect of diamond magnetometry).
When in this review is referred to a specific size FND, this is mentioned in subscript (e.g.
FND70 refers to FNDs with 70 nm diameter) and if not further specified, when ‘NV-centers’ is
mentioned, we usually talk about negatively charged NV-centers. The diamonds used in this group for diamond magnetometry are mechanically grinded, high pressure high temperature (HPHT) produced diamonds.
6
Towards applied diamond magnetometry
Article overview
Table 1. Overview of a set of articles published by the bioanalysis research group
16 19
18
3,10 17
4
Author Title Keywords
Schirhagl, R et al.
(2014)
Nitrogen-Vacancy Centers in Diamond:
Nanoscale Sensors for Physics and Biology10
Diamond synthesis, principle, ODMR
Hemelaar, S. R.
et al. (2017)
Nanodiamonds as multi-purpose labels for microscopy16
Correlative microscopy, imaging setup, uptake, HeLa,
macrophage, Bio-physics Hemelaar, S. R.
et al. (2017)
The interaction of fluorescent
nanodiamond probes with cellular media17
Aggregate (prevention), proteins/salt analysis, DMEM, HeLa, (bio-)chemistry
Hemelaar, S. R.
et al. (2017)
Generally Applicable Transformation Protocols for Fluorescent Nanodiamond Internalization into Cells18
Internalization, Electrical- /chemical transformation, yeast, chemistry
Ong, S. Y. et al (2017)
Shape and crystallographic orientation of nanodiamonds for quantum sensing3
Fundamental, sensitivity, physics
Diamond magnetometry
Medium interaction Cellular uptake &
internalization protocols Imaging technique
& sensitivity
Diamond shape
& properties
Surface functionalization
Figure 3. A systematic overview on different aspect of diamond magnetometry
7 Nitrogen-Vacancy Centers in Diamond: Nanoscale Sensors for Physics and Biology10
This article is a review giving a clear insight into the nanodiamond particle itself and its rough properties. Nanodiamonds can be produced in a variety of ways. Research in the past 10 years, has found detonation diamonds (DNDs), which are created through controlled detonation of explosives.
These DNDs have revealed functionalizable, biocompatible, ~4 nm nanodiamonds with a single fluorescent impurity.20 Although usually many more fluorescence NV-centers are needed for practical usage. Also, these diamonds are aggregating heavily and have a high number of said ‘sp2-like carbon layers’21, which interfere with the fluorescence of the NV-center.
The diamonds which are practical to use for diamond magnetometry, are mechanically grinded diamonds down to tens of nanometers, with a commercial smallest size of ~15 nm. These diamonds are much purer than DNDs, but are relatively large and barely contain NV-defects (for reference 25 nm diamonds contain a NV center in 1/1000 particles).10 But High-Pressure, High-Temperature (HPHT) annealing in combination with high-energy irradiation, creates a large number of NV-defects, so that on average every FND25 host 10~15 NV-centers and exponentially increasing with diamond size.
Usually diamond have oxygenated surfaces, which make them hydrophilic22, but surface modification is easily done as mentioned later in this review.4 Figure 4 shows a wide variety of possible surface functionalization. This is something useful when NDs are used as biolabel, because navigators like biomolecules (e.g. antibodies23, DNA24, etc.…) can be covalently attached, which is one of the strongest bonds between molecules. Electrostatic binding is an easier approach, but since the bonding is non-specific and quite weak this isn’t the preferred option.25
Now knowing a bit about the properties of -our- fluorescent nanodiamonds, the next paper will discuss an actual analysis of internalized FNDs. Showing relatively small (for diamond magnetometry) FND internalization in macrophages and biotin (a ‘steering-protein’) coated FND internalization in Epithelial cells.
Figure 4. Possible surface functionalization’s of nanodiamond surface. By reference 4
8 Nanodiamonds as multi-purpose labels for microscopy16
Diamond magnetometry is a form of correlative microscopy, meaning that a comprehensive view of a sample is made by combining information of different methods of microscopy.26 A common used form of correlative microscopy is CLEM (Correlative light and electron microscopy), but as mentioned in the introduction, this method encounters limitations such as unstable fluoresce probes, large resolution gap or probes not surviving sample preparation.27 This is where FNDs may provide a solution.
Electron microscopy with FNDs has been done in living cells.28,29 However, this has been done with larger FNDs (100-150nm) which limits it to cellular uptake studies only. In this paper it is shown that correlative microscopy is also possible with 40nm and 70 nm FNDs. More precisely, fluorescence Optically Detected Magnetic Resonance (ODMR) and Cathodoluminescence (CL = a property of matter of converting electrons to photons) after internalization is shown. Also, antibody-targeted labeling is done using FND70.
When analyzing the bare particles, the authors found a substantial variation in size for a so said FND size. For FND40 the average size was 67±37nm and FND70 revealed an average size of 54±26nm. To obtain fluorescence, the nanodiamonds must be excited by an optimal wavelength of the incoming laser light. The authors found 561nm to be the optimal wavelength. CL is studied as well, showing a higher intensity for bigger FNDs due to the amount of NV-center per diamond (on average
>300 for FND70 and 10-15 for FND40), but here also a strong variation is included per specific diamond size. Meaning that there are very CL-bright smaller NDs and very CL-dim bigger NDs.
The identity of the FNDs in the samples were determined by their fluorescence and confirmed by the characterizing double dip in the ODMR spectrum (as seen in Figure 2). High resolution localization was done though CLEM overlaid with backscattered electron (BSE)(Figure 5 shows the visualizing of FND70 in epithelial cells, the same was done for FND40 in macrophages, see reference 16). Internalization of FND40 is done in J774 Macrophages. On estimation the macrophages
internalized about 210 FND40 per cell, as expected the fluorescence property remained stable even after osmium fixation, explained by NV-centers embedded in the diamond structure. Internalization with immunolabeling is shown by coating FND70 with streptavidin and coating HT29-EpCAM-GFP cells (a human epithelial colon carcinoma cell line) with biotin is shown successfully (Biotin and streptavidin have a uniquely high affinity for each other and have the strongest non-covalent bond known in nature).
This article has shown the possibility of FM after EM preparations and the visualization of FND by CL, EM, BSE and magnetometry. All these samples are done in-vitro meaning that the cells are present in a medium. It is known that nanoparticles can have interactions with certain media. In the next paper this will be discussed.
Figure 5. Multimodal analysis of FND70 immunolabeling of EpCAM-GFP HT29 cells. (A) Streptavidin conjugated FND70 linked to biotin conjugated HT29 (B) Magnetic resonance spectra where taken at the bright spots identified as diamonds. The lower part of the figure shows the spectrum taken at the circled spot. 2,5% is the contrast between the resonance line and the background for one run. (C) CL, SE and the CL overlaid with BSE images of FND70 labeling an HT29 cell cluster at the cell surface. Note that in the right image single FND70 particles are resolved with CL. Bars: (A, B) 25 μm, (C) 1 μm. By reference 16
9 The interaction of fluorescent nanodiamond probes with cellular media17
In vivo living cells require a certain medium to live or grow, but from literature we know that nano particles like FNDs have an interaction with media, causing the FNDs to aggregate.30 Ground breaking work has been done in the nanoscience24, without taking this in account, but if we want to accurately perform diamond magnetometry, it is important to know how FNDs behave in such environment.
The aggregation of FNDs cause a significant increase in hydrodynamic diameter31, this is particularly important in the case of cellular uptake (which is important in diamond magnetometry), since uptake by endocytosis is dependent on size.32 So, two fundamental question arise. 1) How do FNDs interact with medium? And 2) Which salts or proteins play a role in this? This article17 experimentally gives us answers to these questions.
We know from other nanoparticles that they can acquire a so called “protein corona”
consisting of medium proteins31, this corona can influence the nanoparticles hydrodynamic size, surface charge and aggregation behavior33. This also applies to FNDs, yet it never has been studied how cellular media affects FNDs used as a probe in an intra-cellular application.
This paper studies the interaction of FNDs with DMEM (Dulbecco’s Modified Eagle Medium) and different components of the medium (FBS). DMEM is a commonly used medium to culture mammalian cells. To study cellular uptake of FND, HeLa cells were incubated. For observing the response of the FND in DMEM and HeLa cells, several methods were used. Dynamic light scattering (DLS) is used to obtain aggregate size, Scanning Electron Microscopy (SEM) to observe protein assembly around the diamonds, mass spectrometry to find the most abundant proteins in the mixture and X-ray photoelectron spectroscopy to analyze the inorganic components like salts.
To answer our fundamental questions, the paper found that FNDs in the full medium (proteins + salts) indeed aggregate on a noteworthy scale. However, an easy solution to counter aggregation has been presented. By first introducing FNDs to the proteins of the medium (FBS) and then to the full medium, aggregation is minimalized. When further analyzing this, it is found that FNDs in
uncomplemented DMEM (just salts + glucose) aggregate up to 37 times more than in FNDs in FBS (Ø3700nm vs Ø100nm on average) and thus salts in the medium play a major role in aggregation, whereas proteins do in a lesser way. Also, it is found that the FND surface probably have a
selectiveness for certain proteins, because not necessarily the most dominantly present proteins play a role in the aggregation process. Up to 25 proteins were identified taking part in the process, for salts it is found that sodium chloride plays a major part in the aggregation of FNDs. The experiments were performed with FND25 since they approach the size where FNDs can contain workable stable NV- centers. Since aggregation is determined by surface chemistry, it is expected that the results can be extrapolated to larger diamonds.
Now knowing how to avoid aggregation and thus increase the potential for ND uptake. We subsequently can consider the next matter. For some cells such as macrophages nanoparticles are naturally taken up, but if this is not the case, what can be done then? Next study will investigate this question.
10 Generally Applicable Transformation Protocols for Fluorescent Nanodiamond Internalization into Cells18
Internalization of FNDs into cells is usually not a spontaneous process. When this is not the case a limited number of options are available. In case of HeLa cells with a small stimulus like
electroporation, they ingest BSA-coated diamonds.34 Also, it is possible to chemically coat the nanoparticles or forcefully inject the FNDs with a silicon wire if the cells are big enough (e.g.
oocytes).35 Of said techniques none have been tested on non-mammalian cells. This paper studies internalization techniques on Saccharomyces cerevisiae since their relevance in research. The relevance lies in their ease of genetic modification, outstanding turnover rate, many similarities in DNA compared with human DNA and the unique property of been able to observe their chronological aging.36 This last property is especially interesting, because we now could study DNA damage
associated with aging, through diamond magnetometry (Because DNA damage is associated with radical production).
But in comparison to mammalian cells, yeast have a very thick cell wall which is a barrier to overcome. The authors of this paper study uptake through chemical and electrical transformation. This is observed with confocal microscopy (CM) and sectioning of embedded samples, Colony Forming Units (CFU) is used to assess cell viability (To assess viability, the cells are incubated with the matter to be tested and incubated overnight, to check if the cells still survive/divide properly). Due to light diffraction limits the difference between a single FND or an aggregated clump couldn’t be
differentiated, so the exact amount of FNDs couldn’t be determined. The amount of said ‘objects’ (a single or a cluster FNDs) can be determined and an estimation of particles can be made through an intensity analysis (by an author made FIJI script, FIJI is an image processing program).
Chemical transformation
Chemical transformation means that the respective cells are exposed to a certain chemical, which increases their permeability, so that nanodiamonds can enter the cell more easily. The chemical mixture used is ‘66.6% w/v PEG4000 and 1 M lithium acetate (LiAc). TMIX, FND70, DMSO 5% and 1 M sorbitol’. To not make an overestimation of FNDs the cells were washed with Triton 0.01%, washing away non-internalized FNDs. A quantitative uptake analysis through CM shows an estimated number of particles of ~80 before washing and ~60 after washing. A quantity of near zero objects and particles is revealed for the control. The CFU count showed no significant difference for the complete chemical mixture in comparison to a control group. SEM visualization doesn’t show immediate cellular damage.
Electroporation
The essence of electroporation is permeabilization of the cell membrane, by applying electric pulses to the cells. The quantitative analyses with CM clearly reveal a reduced uptake (up to ¼ of control). The authors give reduced viability and washing away of the FNDs in the electroporation protocol as a possible reason for this result. The CFU count confirmed this result, showing up to a factor 102 reduced viability. SEM visualization further strengthened this by showing a damaged cellular structure.
Bioanalysis show in this paper as first how forced ND internalization can be done when a cell has a thick cell wall. This is most effective and least harmful done through chemical transformation of the respective cell. Until now, it is discussed how to recognize FNDs, how to internalize them and how they behave in-vitro. The next paper is a more fundamental research, discussing in detail the shape of the diamond, which is good to keep in mind for all previous papers and very crucial when diamond magnetometry is to be performed.
11 Shape and crystallographic orientation of nanodiamonds for quantum sensing3
In the past a simplistic spherical model of FNDs were used in the field of quantum sensing, to interpret or predict results37,38. This was done without thoroughly investigation or accurate data. But to
accurately perform diamond magnetometry and interpret acquired data well, it is important to also take in account the actual geometry of such nanoparticle. The author of this paper focusses on a detailed analysis of the geometry of a FND, by performing High Resolution Transmission Electron Microscopy (HR-TEM), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). The FNDs which are studied in this paper are FNDs with a median hydrodynamic diameter of 25 nm
(Microdiamant AG MSY 0.00–0.05), because these are to be used commonly for diamond magnetometry.
SEM is used to determine the lateral dimensions of the FND, 85 particles were analyzed resulting in an average width of 23.2 nm (SD of 11.1 nm). AFM is performed to determine the vertical dimension of the particle, the author found an average vertical component of 4.5 nm (SD of 3.5 nm).
Thus, it can be concluded that the diamonds have a flake like structure. Knowing that NV-center don’t reside within 2 nm of the FND borders37,39, this quickly results in a reduced number of defects, due to the limited height of the crystal.
The sensitivity of a NV-center is highly dependent on its location in respect to the surface of the particle. A theoretical calculation has been made by the author of T1 and T2 times in comparison to the location of the NV-center in the particle and the shape of the particle. Figure 6 shows the theoretical prediction.
The high aspect ratio (about 5) this flake structure has, also influences the uptake of the particle40. A strong indication is found that these particles can puncture and escape endosomes in the cell, whereas diamonds with a rounder structure tend to stay in these endosomes and get excreted by the cell41. The aspect ratio also influences the surface area and certain facets of a diamond crystal are more reactive than others42. Thus, it is important to take this in account when surface chemistry is being modified. Knowing the effect of the shape of a nanodiamond on multiple aspect of diamond magnetometry, is another step forward to applied diamond magnetometry knowing the exact shape of our FNDs.
Figure 6. (a) Calculated 1/T1 and 1/T2 plotted against increasing aspect ratio of a tetragonal model nanodiamond, showing lower values and spread for a flake-like particle compared to the spherical model. (b) Nanodiamonds were modelled in a tetragonal shape with the (110) direction along the z-axis and in a spherical shape as used in reference 11.
Figure by reference 2
12
Discussing and future perspectives
Based on the research of the bioanalysis research group, an all-round basic knowledge on
nanodiamonds is acquired. HPHT, electron irradiated mechanically grinded bulk diamonds prove to be the most useful fluorescent diamond for imaging. When cells do not readily take up the nanoparticles, chemical transformation on the respective cells show the most successful uptake promotion. To further control and steer the nanodiamonds in cells, surface modification can be a viable solution. Through different processes, nanodiamonds surface can easily be modified. Nanodiamonds are experimentally shown to be excellent biocompatible, stable probes, even surviving osmium fixation, where commonly used probes usually lose their fluorescence. After cellular uptake, optically detected resonance and cathodoluminescence is shown. To actually perform diamond magnetometry preliminary research has been done, about the shape and crystal structure of the nanodiamond, which is crucial to know in order to accurately acquire the T1 and T2.
Currently a wide variety of applications is to be explored, think about nanodiamonds aiding in radiology and labeling for diagnostic/therapeutic goals. Another emerging application is ‘scanning magnetometry’, which basically is a nanodiamond applied in an atomic force microscope, by embedding a 2~10 nm nanodiamond in the sensing tip (Figure 7), very high resolution could be acquired through resonance of surface electron spins. But currently it is difficult to prepare the
equipment and embedded sizes below ND10. Also, a new
laser pulsing protocol is currently in development, which plausibly lead to differentiation in for example chemical shifts43, which can result in fast chemical analysis, for example increasing medicine research speed. Very important research is further done towards increased sensitivity as ultimate goal atomic resolution.43 The current sensitivity is already impressive, but it is far from the theoretical possible. To use nanodiamonds as an effective probe, further research is done towards smaller, brighter nanodiamonds.44,45 The problem with smaller, brighter nanodiamonds is due to the fact that NV- centers currently cannot exist within 2 nm of its particle border. This is probably because of unstable charge (in the border area) causing NV- to convert to NV0 (which isn’t magnetic thus a useless NV-center for magnetometry). To go smaller means to find a way to stabilize NV- centers further. As final mention, nanodiamond prove to contribute to medical sciences, even when its full potentials isn’t met yet, but to practically
apply NDs in vivo research should be done beforehand, but this is something which stands in the far future. The bioanalysis research group will for now focus on studying and refining its applicability and actually making diamond magnetometry work in living cells.
Acknowledgments
I would like to thank Romana Schirhagl for the opportunity to let me work in her group and her effort to try to find what really interest me. Furthermore, I like to thank Yori Ong for his flexibility, feedback and steering me in the right direction when needed.
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