Cell imitation and methods for producing it

Abstract

In various embodiments, a cell imitation is provided, comprising a spherical material structure with a diameter of at least 3 µm, wherein the material comprises magnesium fluoride. Furthermore, an ensemble of cell imitations is provided, as well as their use for replicating the optical behavior of cells in an imaging technique. A method for producing a spherical material structure, preferably with a diameter of at least 3 µm, is also provided.

Description

[0001] The present invention relates to a cell mimic, in particular a cell mimic for use as a test standard in a marker-free imaging method. Furthermore, the invention relates to a method for producing the same.

[0002] Laboratory and imaging procedures for GLP / GMP work, diagnostics, regulatory issues, and cross-laboratory collaborations require standards for validation and calibration. In marker-free imaging and flow cytometry, no permanent test standards for validation and calibration are currently known.

[0003] Reliable calibration and validation of marker-free imaging techniques is currently not possible. In marker-free imaging, there are currently no permanent test standards that can be used for calibration, optimization, and testing in quantitative phase imaging (QPI), optical coherence tomography (OCT), flow cytometry (FCM), and flow cytometry imaging under highly realistic measurement conditions. This lack of calibration standards hampers the use of innovative marker-free imaging techniques in industrial and medical applications.

[0004] Currently, there is no known synthetic method for producing monodisperse MgF2 beads with a diameter larger than 2 µm. The scientific article by Xu et al. (1) reports that micro-sized spherical particles with a broad size distribution between 200 nm and 2 µm have been prepared. Typical known synthetic routes for the preparation of magnesium fluoride nanoparticles and microspheres include precipitation, the ammonia route, sol-gel via hydrofluoric acid (HF) or acetate, microwave-assisted ionic liquid solvothermy, sonification (treatment with ultrasonication), and seawater synthesis. Precipitation can be used to produce uniform magnesium fluoride particles with different morphologies by varying the pH and ionic strength while avoiding complexation of interacting ions.Spherical, cubic, prismatic, and platelet-shaped colloidal particles are formed by diffusion growth or aggregation of individual particles. However, it is difficult to control the particle size for particles larger than 2 µm (1, 2, 3).

[0005] The synthesis using ammonia from the reaction of Mg 2+ and F - in aqueous solution can be used to obtain particles with sizes from approximately 6 nm to approximately 300 nm and morphologies from spherical to cubic, which can be produced by a suitable F:Mg ratio (2).

[0006] Sol-gel synthesis using HF requires an inert atmosphere under argon gas and a Schlenk apparatus. The particle purification process is lengthy and requires large amounts of alcohol as a solvent. The resulting size distribution is between 10–20 nm (4). The sol-gel method using acetate is also not suitable for large-scale synthesis. This is due to the release of large amounts of hydrogen during the first step of the reaction between magnesium and methanol (5).

[0007] A redesign of the synthesis using commercially available magnesium ethoxide as starting material is also unsuitable for the intended purpose, as it is insoluble in methanol or ethanol (5). Therefore, transparent sols are not available, as a protective MgF2 layer deposits on the suspended solid Mg(OEt)2 on the precursor particles (5).

[0008] An alternative route using MgCl2 as the starting material is quite simple, but suffers from the stoichiometric formation of HCl from MgCl2 as a result of the reaction with HF (5,6). HCl is highly corrosive, and practical application of such sols is not recommended. However, the synthesis route via ionic liquids can be used as a replacement for a fluorine source instead of working with large amounts of HF. Sonification has so far only been reported for the production of active MgF2 biofilms (7); a corresponding application for the synthesis of particles is not possible and therefore unknown.

[0009] Finally, synthesis using seawater has been described as a cost-effective method (8). However, the use of seawater for industrial synthesis is limited by environmental concerns. Furthermore, the effects of other chemicals in seawater have not yet been fully understood and investigated with regard to how they influence particle size distribution.

[0010] Doping MgF2 beads with activators such as lanthanides is a useful step for further functionalizing the beads. This was demonstrated by Cortelletti et al. for small CaF2 and SrF2 particles as well as for bulk materials (9).

[0011] Based on this known state of the art, the present object can be seen in providing MgF2 particles that emit light preferentially in the visible or near infrared range and can be used as photostable dye-free markers, which collectively mean label-free markers.

[0012] According to the invention, a cell imitation is provided which has a spherical material structure with a diameter of at least 3 µm, wherein the material comprises magnesium fluoride.

[0013] A cell imitation according to the invention can be understood as a particle for imitating a cell, i.e., a particle in which at least one property, in particular an optical property and a size property, corresponds to that of a somatic cell. In this respect, the cell imitation can also be referred to as a cell phantom. Due to its properties, the cell imitation can be used to calibrate and / or adjust an optical measuring system. The spherical material structure can, in particular, comprise a substantially spherical particle.

[0014] The particle according to the invention can be configured to mimic the size and optical properties of somatic cells. An ensemble of particles according to the invention can advantageously be used for calibration, as well as for checking and testing system performance and its optimization under the most realistic measurement conditions possible in quantitative phase imaging (QPI), such as those provided by digital holographic microscopy and related techniques, such as optical coherence tomography (OCT), as well as in marker-free flow cytometry (FCM) and associated flow cytometry imaging methods. This use of at least one cell mimic described here, in particular an ensemble, can occur within the framework of one of these methods.

[0015] The use of the cell mimic according to the invention enables the calibration and validation of label-free measurement systems such as quantitative phase microscopes, tomographic phase microscopy systems, high-resolution OCT systems, and flow cytometers for the analysis of living cells under highly realistic conditions. Due to the longevity of the cell mimic, it can be used repeatedly over a long period of time, allowing an initially adjusted or calibrated measurement system to be monitored and readjusted for possible changes in its operating parameters.

[0016] The spherical structures according to the invention, preferably spheres, comprise magnesium fluoride (MgF2) and combine all the requirements for label-free imaging standards. They are round or spherical, have dimensions in the range of cell sizes, exhibit good durability, and have a refractive index in the range of the cellular refractive index. The spherical material structures according to the invention enable the calibration and validation of label-free measurement systems for the analysis of living cells under highly realistic measurement conditions. The invention uses magnesium fluoride as a durable material with a refractive index and size properties that approximate those of living cells and tissue.Furthermore, based on the present invention, in combination with suitable embedding materials, it is possible to produce novel, permanent tissue phantoms with optical properties (particularly refractive index) and size ratios that essentially correspond to those of living biological specimens. According to one embodiment, the cell imitation consists of magnesium fluoride.

[0017] Another part of the present invention relates to a method for producing or synthesizing cell-sized magnesium fluoride particles. Magnesium fluoride is currently only available as a bulk material or as a material and in the form of nanoparticles. Implementing the present invention and providing magnesium fluoride particles as cell standards requires the synthesis of spherical particles in the size range of 5 µm to 15 µm. The produced particles can be functionalized with fluorescent dyes or doped with luminescent species, which enables broader use, including for fluorescence-based imaging methods.

[0018] The present invention offers numerous advantages. For example, the cell imitations according to the invention can be used in optical measurement systems to calibrate / validate / optimize them using durable test standards. The cell imitations used for this purpose can preferably have optical properties and size characteristics that correspond to those of living cellular samples, thus enabling highly realistic conditions to be created. Cell imitations with these properties can contribute significantly to more reliable system characterization and calibration, more effective system configurations, sophisticated alignments within the overall system, and improved system validation. Furthermore, the cell imitations according to the present invention can contribute to increasing measurement accuracy, which is particularly necessary for exploring and developing new application areas and markets.The new calibration and validation standards based on the cell mimics disclosed herein open markets for clinical diagnostics and industrial applications for quantitative phase imaging, tomographic phase microscopy, OCT, flow cytometry, and imaging flow cytometry.

[0019] According to further embodiments of the cell imitation, the material structure can have a refractive index in the range of approximately 1.3-1.4, preferably in the range of approximately 1.35-1.36. Such a choice of refractive index is advantageous because it lies in the range of the refractive index of somatic cells and confers on the cell imitation according to the invention the ability to mimic somatic cells with regard to their optical properties.

[0020] According to further embodiments of the cell imitation, the spherical material structure may have a doping comprising a fluorescent and / or luminescent material.

[0021] According to further embodiments of the cell imitation, the material structure can have a diameter in the range of about 5 µm to about 15 µm.

[0022] According to the invention, an ensemble of cell imitations is further provided in which at least 99% of the cell imitations have a diameter that deviates by a maximum of 5% from the mean diameter. In other words, the ensemble can comprise a monodisperse plurality of cell imitations, which thus exhibits a substantially homogeneous optical behavior.

[0023] The invention further provides a use of the cell mimic described herein, preferably an ensemble of cell mimics, for reproducing or simulating the optical behavior of cells in an imaging method. The cells whose behavior is to be simulated can, in particular, be somatic cells.

[0024] According to one embodiment of the use according to the invention, the imaging method can be quantitative phase-contrast imaging or marker-free flow cytometry. In principle, the imaging method can be any imaging method, for example, one of the methods already mentioned. Imaging can be based on electromagnetic radiation in the visible wavelength spectrum or at other wavelengths, such as in the IR or UV range.

[0025] According to one embodiment of the use according to the invention, this can occur during a calibration process and / or a functional verification test of the device performing the imaging method. The use can occur as part of a first calibration or a first functional verification, or repeatedly for the purpose of optimizing the process.

[0026] According to the invention, there is further provided a method for producing a spherical material structure, preferably with a diameter of at least 3 µm, wherein the material comprises magnesium fluoride, the method comprising the following steps: (i) mixing magnesium nitrate and sodium dihydrogen phosphate in water; (ii) adding an acid to dissolve the salt precipitated in step (i); (iii) adjusting the pH to about 6 to about 8; (iv) heating the mixture to a maximum of 45°C; (v) adding a fluoride source and mixing at a maximum of 45°C; and (vi) heating the mixture in an autoclave at about 150°C to about 250°C to obtain the spherical material structure.

[0027] According to one embodiment of the process according to the invention, the magnesium nitrate may be Mg(NO3)2*6H2O and / or the sodium dihydrogen phosphate may be NaH2PO4*2H2O.

[0028] According to one embodiment of the process according to the invention, the concentration of magnesium nitrate and sodium dihydrogen phosphate can each be about 0.05 mol / L to about 2.0 mol / L, preferably about 0.13 mol / L each.

[0029] According to one embodiment of the process according to the invention, in step (i) a lanthanide nitrate can further be mixed with the magnesium nitrate and the sodium dihydrogen phosphate in water.

[0030] According to one embodiment of the process according to the invention, the amount of lanthanide nitrate may be about 0.01 mol% to about 20 mol%, preferably about 1 mol%, in each case based on a total amount of lanthanide nitrate and magnesium nitrate of 100 mol%.

[0031] According to one embodiment of the process according to the invention, the lanthanide nitrate can be europium nitrate, preferably Eu(NO3)3*2H2O, or terbium nitrate, preferably Tb(NO3)3*2H2O.

[0032] According to one embodiment of the process according to the invention, in step (ii) the acid may be hydrochloric acid (HCl).

[0033] According to one embodiment of the process according to the invention, in step (iii) the pH can be adjusted to about 6.5 to about 7.0.

[0034] According to one embodiment of the process according to the invention, steps (i) to (iii) can be carried out at room temperature (preferably about 15°C to about 25°C).

[0035] According to one embodiment of the process according to the invention, the mixture in step (iv) can be heated to a maximum of 45°C, preferably to a maximum of 40°C, and even more preferably to approximately 37°C. At too high a temperature, the associated reaction can be less well controlled, and the size distribution becomes broader. From this perspective, a maximum temperature of 45°C is advantageous. At temperatures below 40°C, e.g., at 37°C, a relatively homogeneous size distribution is obtained even with uneven heat transfer.

[0036] According to one embodiment of the process according to the invention, in step (iv) the heating of the mixture can be carried out at a rate of about 5°C / min to about 20°C / min, preferably at a rate of about 10°C / min.

[0037] According to one embodiment of the process according to the invention, step (iv) can be carried out in an autoclave.

[0038] According to one embodiment of the process according to the invention, the addition of the fluoride source and mixing in step (v) can be carried out at a maximum of 45°C, preferably at a maximum of 40°C, and even more preferably at approximately 37°C. Regarding the selected temperatures, the same applies as for the previously explained temperature selection when heating the mixture in step (iv).

[0039] According to one embodiment of the process according to the invention, the fluoride source may be an ionic liquid, preferably 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM(BF4)]).

[0040] According to one embodiment of the process according to the invention, the heating in step (vi) can be carried out at about 200°C. When carrying out the process in a tightly sealed container, e.g., an autoclave, the pressure can be in the range of about 150 bar to about 250 bar, preferably in a range of 175 bar to about 225 bar, and more preferably at about 200 bar.

[0041] According to one embodiment of the process according to the invention, the heating in step (vi) can be carried out for about 1 day to about 6 days, preferably for about 4 days.

[0042] According to one embodiment of the process according to the invention, this may further comprise cooling the mixture after step (vi), for example to room temperature.

[0043] According to one embodiment of the process according to the invention, this may further comprise isolating the obtained spherical material structure, preferably by centrifugation.

[0044] According to one embodiment of the method according to the invention, this may further comprise washing the spherical material structure, preferably with water and / or ethanol.

[0045] According to one embodiment of the method according to the invention, this may further comprise dispersing the spherical material structure in water.

[0046] According to one embodiment of the method according to the invention, this may further comprise drying the spherical material structure.

[0047] According to one embodiment of the method according to the invention, the spherical material structure can be defined as in one of the previously described embodiments.

[0048] The features mentioned above and those to be explained below can be used not only in the combination specified in each case, but also in other combinations or on their own, without departing from the scope of the present invention.

[0049] Further advantages and embodiments of the invention will become apparent from the description and the accompanying drawings. Fig. Figure 1 shows energy dispersive X-ray spectroscopy data of the composition of the cell mimics according to the invention. Fig. 2A-2C show a scanning transmission electron microscopy image and the reconstruction of the spatial localization of magnesium and fluorine from spatially resolved energy-dispersive X-ray spectroscopy data of undoped cell mimics. Fig. 3 shows a field emission scanning electron microscope image of the cell mimics according to an embodiment of the present invention. Fig. 4 shows one excitation and three emission spectra of europium-doped cell mimics according to an embodiment of the present invention. Fig. 5 shows excitation spectra at two different excitation wavelengths of europium-doped cell mimics according to an embodiment of the present invention. Fig. 6 shows emission spectra of terbium-doped cell imitations according to an embodiment of the present invention. Fig. 7 shows an excitation spectrum of terbium-doped cell mimics according to an embodiment of the present invention.

[0050] In Fig. Figure 1 shows an energy-dispersive X-ray spectrum of a sample recorded with a scanning transmission electron microscope (STEM-EDX). The x-axis represents the energy in kiloelectronvolts, and the y-axis represents the intensity of the recorded X-ray signal in arbitrary units. The sample on which the X-ray analysis is based consists of a large number of particles or spheres of pure magnesium fluoride. The recorded signal confirms the presence of magnesium and fluoride, with the ratio being 35.51 wt.% for magnesium to 60.05 wt.% for fluoride (based on atomic weight). The remaining 4.44 wt.% is accounted for by carbon and copper, which are contained in the TEM grid on which the sample is placed during the examination.The carbon signal, whose maximum is at 0.28 keV, is obscured by the strong fluorine signal at 0.68 keV and is therefore not visible in the diagram. The two copper signals originate from two different copper transitions (Cu-Kα at 8 keV and Cu-Kβ at 8.9 keV).

[0051] In the Fig. 2A-2C, the spatial structures of the cell mimics were reconstructed from data obtained using a scanning transmission electron microscope combined with an energy-dispersive X-ray spectrometer. The sample consists of a large number of cell mimics made of pure magnesium fluoride. Fig. 2A, the spatial structure of the cell mimics has been reconstructed from the cumulative response signal. In Fig. 2B, the spatial structure of the cell mimics has been reconstructed from the magnesium-derived response signal and in Fig. In Figure 2C, the spatial structure of the cell mimics has been reconstructed from the response signal originating from fluoride. Comparing the reconstructions, it can be seen that the response signals originate from the magnesium atoms ( Fig. 2B) and the response signals from the fluoride atoms ( Fig. 2C) are colocalized. Overall, it can also be seen that the cell mimics are spherical material or substance structures.

[0052] In Fig. Figure 3 shows another image of an ensemble of cell mimics, taken with a field emission scanning electron microscope. The sample examined contains spherical particles of pure magnesium fluoride with an average diameter of 5.2 µm ±0.2 µm.

[0053] In Fig. Figure 4 shows an emission spectrum and three excitation spectra of europium-doped cell mimics. The lower x-axis represents the wavenumber in 1 / cm and the upper x-axis the corresponding wavelength in nanometers. The y-axis represents the normalized intensity in arbitrary units (au). The emission spectrum, whose discrete peak lies at approximately 600 nm, shows the emission at an excitation wavelength of 389 nm (25710 cm -1 ). The excitation spectra were measured for emission wavelengths of 720 nm (13890 cm -1 ), 615 nm (16260 cm -1 ) and 597 nm (16750 cm -1 ) by cell imitations made of magnesium fluoride, which were treated with 10% Eu 3+ have been doped (MgF2: 10% Eu 3+ ). Observing these signals allows the presence of europium in the oxidation state +3 to be confirmed.

[0054] In Fig. Figure 5 shows emission spectra at two different excitation wavelengths of europium-doped cell imitations according to an embodiment of the present invention. The axis labels of the diagram correspond to those of Fig. 4. By comparing the emission spectra in Fig. 5 shows that the emission in the red wavelength range at an excitation wavelength of 389 nm (25710 cm -1 ) is significantly stronger than with short-wave excitation (262 nm).

[0055] In Fig. Figure 6 shows an emission spectrum of cell imitations doped with 10% terbium (MgF2: 10% Tb 3+ ). The sample was irradiated at a wavelength of 350 nm (28570 cm -1 ) was suggested. As in Fig. 4, the lower x-axis represents the wavenumber in 1 / cm and the upper x-axis the corresponding wavelength in nanometers. The y-axis represents the normalized intensity in arbitrary units (au). The strong emission in the green wavelength range is characterized by a distinct energy band at 544 nm (18380 cm -1 ) conditional.

[0056] In Fig. 7 is finally an excitation spectrum of a sample at an emission wavelength of 544 nm (18380 cm -1 ), i.e. in the green wavelength range. As shown in the Fig. 4 and Fig. 6, the lower x-axis represents the wavenumber in 1 / cm and the upper x-axis the corresponding wavelength in nanometers. The y-axis represents the normalized intensity in arbitrary units (au). The sample is, as in the sample in Fig. 6, to produce cell imitations doped with 10% terbium (MgF2: 10% Tb3+ ). From the excitation spectrum, one can see that it is possible to excite the terbium-doped cell mimics in approximately between 250 nm (40000 cm -1 ) and 300 nm (33330 cm -1 ) to produce strong emission in the green wavelength range.

[0057] The above-described particles according to the invention are highly suitable for label-free imaging using digital holography-based quantitative phase imaging (QPI) and tomographic phase microscopy. From the examination of QPI images of the particles according to the invention, it can be concluded that the size and refractive index properties of the particles are similar to those of living somatic cells (diameter 5 µm, refractive index 1.35-1.36). In summary, the results demonstrate that spherical magnesium fluoride particles can be produced with a size and refractive index similar to those of somatic cells and are therefore highly suitable as permanent reference materials for QPI measurements and new, application-specific calibration methods in flow cytometry.

[0058] The particles according to the invention can be produced by using a special mechanism during their synthesis, which allows improved control over the growth of MgF2 particles. In particular, spherical MgF2 particles with a diameter of 5 µm and larger can be produced. The invention comprises a novel synthesis route with at least three possible routes for the production of monodisperse MgF2 particles with diameters greater than 5 µm.

[0059] As shown above, doped MgF2 particles, such as MgF2:Xn 3+ (where Xn=Eu / Tb) can be produced that can emit in the visible range of the electromagnetic spectrum, whereby the dopant is by no means limited to the Eu or Tb ions mentioned.

[0060] The spherical MgF2 particles used as samples, with a diameter of approximately 5 µm, can be produced by hydrothermal synthesis in an autoclave as a reaction vessel in an ionic liquid. For example, 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM(BF4)] can be used here. Highly pure 99.9% Mg(NO3)2*6H2O, NaH2PO4*2H2O, Eu(NO3)3*2H2O, and Tb(NO3)3*2H2O were used as reactants.

[0061] Through the systematic use and variation of supersaturation, pH, temperature, pressure, and time, the reaction rate and particle growth can be controlled. The particle size can be reduced to a diameter of 5 µm or more (i) in a microwave or (ii) in a hydrothermal autoclave, or (iii) by a combination of both methods. The microwave process is faster but requires several steps and is more expensive. The process in a hydrothermal autoclave is simple and typically yields 0.50 g of material per batch. The result is spherical particles that form by self-assembly of MgF2 precursors in the form of nano-whiskers.

[0062] The optional doping process with Nn 3+ involves a two-step mechanism: 1) in situ hydrolysis of the formed XnPO4 salts and 2) adjustment of the pH of the resulting solution to 6.5 and 7.0 before heating.

[0063] An exemplary course of a synthesis by means of which the particles according to the invention can be produced can comprise the following steps: i) Prepare a 0.13 M solution of Mg(NO3)2*6H2O and NaH2PO4*2H2O in 8.75 mL of water. The water can preferably be filtered using a filter (e.g., a Teflon filter) with a pore size of 0.2 µm. ii) Gradually add HCl until the precipitated salt has dissolved. iii) Optionally adjust the pH to approximately 6.5-7.0. iv) Mixing the solution in a reactor, preferably an autoclave, which serves as the reactor for the hydrothermal synthesis. The internal volume can be, for example, 75 mL. v) Heating the solution in the reactor to a maximum of 37°C (preferably at 10°C / min) to induce nucleation of magnesium fluoride. vi) Add 3 mL of an ionic liquid (preferably 1-butyl-3-methylimidazolium tetrafluoroborate) to the solution while stirring at approximately 37°C until a homogeneously cloudy solution is obtained. vii) Preheat the reactor to 50°C for 30 minutes on a hotplate, then place the reactor in a preheated oven and heat at a temperature of approximately 200°C for four days. The specific design of this step is optional and can be carried out using other parameters. The formation of the particles according to the invention begins during this step. The pressure within the reactor plays an important role in the growth of the particles up to a size of approximately 2.5 µm. The synthesis takes place in a closed reactor, so the pressure does not have to be measured directly in the chamber, but can be determined indirectly based on the known internal volume of the reactor in combination with the choice of temperature. viii) Rapidly transfer the contents of the reactor to a centrifuge after it has cooled to room temperature. ix) Centrifuge the cooled contents of the reactor in a centrifuge (preferably at 6000 rpm for 5 minutes). x) Decant the supernatant and add 11 mL of ethanol. xi) Repeat centrifugation three times with ethanol and then twice with water. xii) Disperse the particles in 11 mL of water. xiii) In an optional step, the substance remaining after the centrifugation processes can be dried. This can be done, for example, preferably at atmospheric pressure at 60°C for 1 h.

[0064] If the particles according to the invention are to be enhanced with the property of photoluminescence, a microwave step can preferably be used to dope the starting material (MgF2) with lanthanides in a preliminary step. Thus, doped nano-whiskers are first produced using a microwave. These nano-whiskers are then mixed with the reactants in a second reaction after a washing step and introduced into the reactor for further reaction. A shell then grows around the whiskers produced in the first reaction. Overall, this can enable photoluminescence or, depending on the degree of doping, enhance it.

[0065] To obtain funding, for example, with Eu 3+ / Tb 3+of the starting material, the stoichiometry of Mg(NO3)2*6 H2O with Eu(NO3)3*2 H2O or Tb(NO3)3*2H2O can be adjusted after step i) above, for example in a range between a few percent to about 20% Eu 3+ / Tb 3+ , for example, 10%. To dissolve the precipitated phosphate salt, HCl can be added dropwise (according to step ii) above). The pH can be adjusted to 6.5 or 7 by adding a few drops of pH 10 buffer (according to step iii) above). The steps above can then be carried out, starting with step iv). Bibliography (1) Xu, J.-S.; Zhu, Y.-J. Microwave-Assisted Ionic Liquid Solvothermal Rapid Synthesis of Hollow Microspheres of Alkaline Earth Metal Fluorides (MF2, M = Mg, Ca, Sr). CrystEngComm 2012, 14 (8), 2630-2634; https: / / doi.org / 10. j039 / C2CE06619J (2) Nandiyanto, A. B. D.; Iskandar, F.; Ogi, T.; Okuyama, K. Nanometer to Submicrometer Magnesium Fluoride Particles with Controllable Morphology. Langmuir 2010, 26 (14), 12260-12266; https: / / doi.org / 10.1021 / la101194w (3) Sevonkaev, I.; Matijevic, E. Formation of Magnesium Fluoride Particles of Different Morphologies. Langmuir 2009, 25 (18), 10534-10539. https: / / doi.org / 10.1021 / 1a901307t (4) Noack, J.; Emmerling, F.; Kirmse, H.; Kemnitz, E. Sols of Nanosized Magnesium Fluoride: Formation and Stabilisation of Nanoparticles. J. Mater. Chem. 2011, 21 (38), 15015-15021; https: / / doi.ora / 10.1039 / C1J M11943E (5) Scheureil, K.; Noack, J.; König, R.; Hegmann, J.; Jahn, R.; Hofmann, Th.; Löbmann, P.; Lintner, B.; Garcia-Juan, P.; Eicher, J.; Kemnitz, E. Optimisation of a Soi-Gei Synthesis Route for the Preparation of MgF 2 Particles for a Large Scale Coating Process. Dalton Trans. 2015, 44 (45), 19501-19508. https: / / doi.Org / 10.1039 / C5DTQ2196K (6) Kemnitz, E. Fluorolytic Sol-Gel Processes. In Handbook of Sol-Ge! Science and Technology; Klein, L., Aparicio, M., Jitianu, A., Eds.; Springer Internationa! Publishing: Cham, 2016; pp 1-41; https: / / doi.ora / 10.1007 / 978-3-319-19454-7 114-1 (7) Leilouche, J.; Friedman, A.; Lahmi, R.; Gedanken, A.; Banin, E. Antibiofiim surface functionalization of catheters by magnesium fluoride nanoparticles https: / / www.dovepress.com / antibiofilm-surface-functionalization-of-catheters-bymagnesium-fluoripeer-reviewed-article-IJN (accessed Apr 14, 2020); https: / / doi.org / 10.2147 / IJN.S26770 (8) Qin, T.; Zhang, P.; Qin, W. A Novel Method to Synthesize Low-Cost Magnesium Fluoride Spheres from Seawater. Ceramics International 2017, 43 (16), 14481-14483; https: / / doi.Org / 10.1016 / i.ceramint.2017.06.145 (9) Paolo Cortelletti, Marco Pedroni, Federico Boschi, Sonia Pin, Paolo Ghigna, Patrizia Canton, Fiorenzo Vetrone, and Adolfo Speghini Crystal Growth & Design 2018 18 (2), 686-694; https: / / doi.ora / 10.1021 / acs.cad.7b01050. QUOTES CONTAINED IN THE DESCRIPTION

[0000] This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions. Cited non-patent literature

[0000] Xu, J.-S.; Zhu, Y.-J. Microwave-Assisted Ionic Liquid Solvothermal Rapid Synthesis of Hollow Microspheres of Alkaline Earth Metal Fluorides (MF2, M = Mg, Ca, Sr). CrystEngComm 2012, 14 (8), 2630-2634; https: / / doi.org / 10. j039 / C2CE06619J

[0065] Nandiyanto, A. B. D.; Iskandar, F.; Ogi, T.; Okuyama, K. Nanometer to Submicrometer Magnesium Fluoride Particles with Controllable Morphology. Langmuir 2010, 26 (14), 12260-12266; https: / / doi.org / 10.1021 / la101194w

[0065] Sevonkaev, I.; Matijevic, E. Formation of Magnesium Fluoride Particles of Different Morphologies. Langmuir 2009, 25 (18), 10534-10539. https: / / doi.org / 10.1021 / 1a901307t

[0065] Noack, J.; Emmerling, F.; Kirmse, H.; Kemnitz, E. Sols of Nanosized Magnesium Fluoride: Formation and Stabilisation of Nanoparticles. J. Mater. Chem. 2011, 21 (38), 15015-15021; https: / / doi.ora / 10.1039 / C1J M11943E

[0065] Scheureil, K.; Noack, J.; König, R.; Hegmann, J.; Jahn, R.; Hofmann, Th.; Löbmann, P.; Lintner, B.; Garcia-Juan, P.; Eicher, J.; Kemnitz, E. Optimisation of a Soi-Gei Synthesis Route for the Preparation of MgF 2 Particles for a Large Scale Coating Process. Dalton Trans. 2015, 44 (45), 19501-19508. https: / / doi.Org / 10.1039 / C5DTQ2196K

[0065] Kemnitz, E. Fluorolytic Sol-Gel Processes. In Handbook of Sol-Ge! Science and Technology; Klein, L., Aparicio, M., Jitianu, A., Eds.; Springer Internationa! Publishing: Cham, 2016; pp 1-41; https: / / doi.ora / 10.1007 / 978-3-319-19454-7 114-1

[0065] Leilouche, J.; Friedman, A.; Lahmi, R.; Gedanken, A.; Banin, E. Antibiofiim surface functionalization of catheters by magnesium fluoride nanoparticles https: / / www.dovepress.com / antibiofilm-surface-functionalization-of-catheters-bymagnesium-fluoripeer-reviewed-article-IJN (accessed Apr 14, 2020); https: / / doi.org / 10.2147 / IJN.S26770

[0065] Qin, T.; Zhang, P.; Qin, W. A Novel Method to Synthesize Low-Cost Magnesium Fluoride Spheres from Seawater. Ceramics International 2017, 43 (16), 14481-14483; https: / / doi.Org / 10.1016 / i.ceramint.2017.06.145

[0065] Paolo Cortelletti, Marco Pedroni, Federico Boschi, Sonia Pin, Paolo Ghigna, Patrizia Canton, Fiorenzo Vetrone, and Adolfo Speghini Crystal Growth & Design 2018 18 (2), 686–694; https: / / doi.ora / 10.1021 / acs.cad.7b01050

[0065]

Claims

[1] Cell imitation, comprising: a spherical material structure with a diameter of at least 3 µm, wherein the material comprises magnesium fluoride. [2] Cell imitation according to claim 1, wherein the material structure has a refractive index in the range of about 1.3-1.4, preferably in the range of about 1.35-1.

36. [3] Cell imitation according to claim 1 or 2, wherein the spherical material structure has a doping comprising a fluorescent and / or luminescent material. [4] Cell imitation according to one of claims 1 to 3, wherein the material structure has a diameter in the range of about 5 µm to about 15 µm. [5] Ensemble of cell imitations according to one of claims 1 to 4, wherein at least 99% of the cell imitations have a diameter which deviates by a maximum of 5% from the mean value of the diameters. [6] Use of a cell imitation according to one of claims 1 to 4, preferably an ensemble of cell imitations according to claim 5, for simulating an optical behavior of cells in an imaging method. [7] Use according to claim 6, wherein the imaging method comprises quantitative phase contrast imaging or label-free flow cytometry. [8] Use according to claim 6 or 7, wherein the use occurs during a calibration process and / or a functional verification test of the device performing the imaging method. [9] A method for producing a spherical material structure, preferably with a diameter of at least 3 µm, wherein the material comprises magnesium fluoride, comprising: (i) mixing magnesium nitrate and sodium dihydrogen phosphate in water; (ii) adding an acid to dissolve the salt precipitated in step (i); (iii) adjusting the pH to about 6 to about 8; (iv) heating the mixture to a maximum of 45°C; (v) adding a fluoride source and mixing at a maximum of 45°C; and (vi) heating the mixture in an autoclave at about 150°C to about 250°C to obtain the spherical material structure. [10] The process according to claim 9, wherein the magnesium nitrate is Mg(NO3)2*6H2O and / or wherein the sodium dihydrogen phosphate is NaH2PO4*2H2O. [11] A process according to claim 9 or 10, wherein in step (i) a lanthanide nitrate is further mixed with the magnesium nitrate and the sodium dihydrogen phosphate in water.

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