Cross Reference to Related Application

This application claims the benefit of U.S. Provisional Application No. 61/877,732, filed on September 13, 2013, the content of which is incorporated herein by reference in its entirety.

Statement Regarding Federally Sponsored Research or Development

This invention was made with Government support under Grant No. FA9550-11-1- 0270, awarded by the U.S. Air Force, Office of Scientific Research and funds from the QuASAR program at the Defense Advanced Research Project Agency (DARPA). The Government has certain rights in the invention.

Background

Nanoparticles have emerged in recent years as a promising approach to particulate labeling probes for multimodal imaging, and also for targeted drug or gene delivery. They can also be used for tracking at the single-molecule level. In general, little is known about the immediate environment of targeted nanoparticles due to a lack of suitable visualization protocols. Knowledge of the local environment would allow clear delineation of relationships between the activity of a labeled agent and the local biological environment. One would like to know, for example, the nature and proximity to adjacent macromolecular assemblies. Despite considerable investigation, the identification and optimization of such a cross- platform biomarker has remained elusive.

Optical microscopy offers versatility, specificity, and sensitivity in fixed and live cell settings. The development of super-resolution techniques has improved spatial resolution to tens of nanometers, but these techniques are restricted to a subset of cellular processes. Transmission electron microscopy (TEM) offers higher spatial resolution. But, it lacks reliable multimodal markers to align the field-of-view with a desired subcellular region. This is particularly true if multiple iterations between imaging techniques is desired. Routine multimodal correlated imaging, which could allow the study of live cells with concurrent visualization of ultra-structural details, would involve the development of inert biomarkers and protocols for correlating optical and electron microscopies.

Reporter systems suitable for both optical and TEM imaging are lacking. For example, fluorescent dyes and fluorophores for fluorescence microscopy cannot be resolved by TEM, can be cytotoxic, and photobleach under optical excitation. Several candidate markers for multimodal imaging have been pursued, but none has yet been found to be practical and universally applicable. Gold nanoparticles and nano-gold cluster compounds are readily bio-conjugated and resolvable by TEM due to their strong electron scattering. Gold can be cytotoxic by itself, and frequently becomes isolated in lysosomes in live cells. Detergent can be used after fixation to allow penetration into cells, but penetration and immunolabeling efficiency can be low, particularly with colloidal gold, due to steric hindrance. Particulate markers such as gold particles are frequently applied after cryo-slicing and thawing, after cryo-fracture, or post-embedding after slicing. That is, after cell death. These approaches have higher immunolabeling success rates, which is dependent upon particle size and whether or not a slice is embedded. While the cryo-slicing approach has produced some impressive correlative imaging results, it is an exceptionally difficult approach, and is also not amenable to multiple iterations between imaging techniques. Last, immunogold without fluorophores can be used as a particulate marker, but for typical particle sizes, involves silver-enhancement to enlarge the particles to render them visible in optical and/or electron microscopies of slices.

Colloidal quantum dot (QD) biolabels are resolvable by TEM, do not photobleach, and can be functionalized with antigen-specific antibodies for targeting. They are, however, cytotoxic, so their surfaces should be decorated with organic conjugants to provide a barrier. QDs also suffer from "blinking" problems and are incompatible with the osmium tetroxide stain frequently used in sample preparation for TEM Summary

Many embodiments described herein relate to a composition, comprising fluorescent nanodiamonds, wherein the nanodiamonds are surface functionalized with (a) a specific binding agent, and/or (b) a transfection agent.

In some embodiments, the nanodiamonds are conjugated to both a transfection agent and a specific binding agent.

In some embodiments, the nanodiamonds are conjugated to a specific binding agent, wherein the specific binding agent is an antibody.

In some embodiments, the nanodiamonds are conjugated to a transfection agent, wherein the transfection agent is a dendrimer.

In some embodiments, the nanodiamonds are further surface functionalized with a therapeutic agent. In some embodiments, the nanodiamonds are further surface functionalized with a diagnostic agent.

In some embodiments, the nanodiamonds comprise negatively charged fluorescent nitrogen-vacancy (NV ^~ ) centers. In some embodiments, the nanodiamonds comprise a fluorophore other than the nitrogen- vacancy (NV ) centers.

In some embodiments, the nanodiamonds have an average size of about 1 nm to about 50 nm. In some embodiments, the nanodiamonds have an average size of about 1 nm to about 500 nm. In some embodiments, the nanodiamonds have an average size of about 1 nm to about 900 nm, or from about 1 nm to about 800 nm, or from about 1 nm to about 700 nm, or from about 1 nm to about 600 nm, or from about 1 nm to about 400 nm, or from about 1 nm to about 300 nm, or from about 10 nm to about 250 nm, or from about 10 nm to about 200 nm, or from about 10 nm to about 150 nm.

In some embodiments, the nanodiamonds are conjugated to a dendrimer via maltotriose. In some embodiments, at least 50% of the surface amine sites on the nanodiamonds, or at least 60% of the surface amine sites on the nanodiamonds, or at least 70% of the surface amine sites on the nanodiamonds, or at least 80% of the surface amine sites on the nanodiamonds, or at least 90% of the surface amine sites on the nanodiamonds, or at least 95% of the surface amine sites on the nanodiamonds, are conjugated to maltotriose in order to eliminate cytotoxicity. In some embodiments, (a) the nanodiamonds comprise fluorescent nitrogen-vacancy (NV ^~ ) centers; (b) the nanodiamonds are conjugated to a dendrimer as a transfection agent; (c) the nanodiamonds are conjugated to an antibody as a specific binding agent; and (d) the nanodiamonds have an average size of about 1 nm to about 500 nm.

In some embodiments, the nanodiamonds are not cytotoxic.

Further embodiments described herein relate to a biological sample transfected with the nanodiamonds composition described above.

In some embodiments, the biological sample comprises a live cell transfected with the nanodiamonds. In some embodiments, the biological sample comprises a sliced or cryo- fractured cell sample transfected with the nanodiamonds.

Further embodiments described herein relate to a method for therapy, diagnosis, or scientific studies in biology, comprising: contacting a biological sample or a biological subject with the nanodiamonds composition described above; and subjecting the biological sample to at least one of an optical microscopy and an electron microscopy. In some embodiments, a biological sample can be obtained from a biological subject that is administered with the nanodiamonds composition, such as an animal or a human subject, and the same biological sample can be subjected to both optical microscopy and electron microscopy. In some embodiments, at least two different biological samples can be obtained from the biological subject, such as at different times or from different locations on the biological subject, and one biological sample can be subjected to optical microscopy, and another biological sample can be subjected to electron microscopy.

In some embodiments, the method comprises subjecting the biological sample to both the optical microscopy and the electron microscopy.

In some embodiments, the contacting step comprises transfecting the biological sample with the composition comprising the nanodiamonds.

In some embodiments, the method comprises exciting the fluorescent nanodiamonds, and detecting the fluorescence signal by optical microscopy.

In some embodiments, the method comprises detecting the nanodiamonds by transmission electron microscopy. In some embodiments, the transmission electron microscopy comprises (a) using a restrictive objective aperture to block diffracted beams from the nanodiamonds, and/or (b) examining electron diffraction patterns to detect the nanodiamonds based on their crystallimty. In some embodiments, the diffraction patterns can be identified in reciprocal space whereas. In other embodiments the signatures of crystallimty can be identified in real space as Fresnel fringes.

In some embodiments, the method comprises delivering to a living cell a therapeutic or diagnostic agent conjugated to the nanodiamonds, and detecting the presence of the nanodiamonds by at least one of an optical microscopy and an electron microscopy.

Additional embodiments described herein relate to a composition comprising nanodiamonds conjugated to dendrimers for facilitating transfection. In some embodiments, the nanodiamonds do not comprise a fluorophore. In some embodiments, the nanodiamonds are conjugated to an antibody for targeting a specific antigen.

Additional embodiments described herein relate to a method for therapy, diagnosis, or scientific studies in biology, comprising: contacting a biological sample with nanodiamonds; and detecting the nanodiamonds by transmission electron microscopy, wherein the transmission electron microscopy comprises (a) using a restrictive objective aperture to block diffracted beams from the nanodiamonds, and/or (b) examining electron diffraction patterns to detect the nanodiamonds based on their crystallimty.

Additional embodiments described herein relate to a method for treating a disease and determining the efficacy and location of treatment, comprising administrating to a patient a therapeutic agent conjugated to nanodiamonds, and determining the location of the therapeutic agent by detecting the presence of the nanodiamonds.

Additional embodiments described herein relate to a method for diagnosing a disease, comprising administrating to a patient a diagnostic agent conjugated to nanodiamonds, and determining the location of the diagnostic agent by detecting the presence of the nanodiamonds.

Brief Description of the Drawings

Figure 1 shows fluorescence lifetime imaging of cells with NDs (top row), with corresponding fluorescence lifetime spectra (bottom row), (a) HeLa cells stained with DAPI. (b) Unstained HeLa cells, (c) NDs incubated with HeLa cells.

Figure 2 shows a comparison of distributions of nanodiamonds in HeLa cells by fluorescence lifetime imaging, (a) Cellular distribution observed in untransfected NDs (control) in excess concentration, (b) Magnified image of an untransfected cell showing nucleus and cytoplasm with aggregated nanodiamonds primarily outside of cells, (c) Uniform distributions of NDs throughout the cytoplasm (no DAPI stain) in NDs transfected into cells with PPI dendrimers. (d) Less uniform distribution of NDs added to cells without transfecting agent (brightness enhanced, no DAPI).

Figure 3 shows TEM characterization of a control sample revealing the typical appearance of NDs in non-transfected HeLa cells, (a) Image of several loose agglomerations of NDs in the extracellular matrix. Cytoplasm (Cy), nanodiamonds (ND). Scale bar, 500 nm. (b) Electron diffraction pattern of the same area, confirming the agglomerations to comprise NDs. The shadow is from a pointer used to block the central beam for image acquisition.

Figure 4 shows TEM verification of ND transfection and adherence to nuclear membrane of HeLa cells, (a) Image of a portion of a cell with a ND adhered to the nuclear membrane. While arrows indicate the nuclear membrane. Stripes are knife marks from the ultramicrotome, and the wide, horizontal dark band is due to a slight wrinkle of the slice. Scale bar, 500 nm. (b) Magnified image of single ND (consisting of three domains). Scale bar, 50 nm. (c) HRTEM image of the ND, showing lattice fringes (indicated by pairs of lines) which verify its crystalline character. Scale bar, 1 nm. (d) Electron diffraction pattern of the same area, further verifying that the particle is a (crystalline) ND. The shadow is from a pointer, used to block the central beam from image acquisition. Cytoplasm (Cy), Nucleus (Nu), nanodiamond (ND).

Figure 5 shows confocal microscopy of nanodiamonds. The threshold for red pseudocolor is set to an identical level for all images (a-d). (a) Confocal image of cells without NDs (control) showing only one small red dot above the selected threshold. This bright dot is not from diamond, but is due to background fluorescence, (b) In-plane (X-Y) confocal image of HeLa cells incubated with NDs. No transfection reagent was used, (c) Y-Z slice reconstruction showing cells between the glass coverslip and slide. The boundaries of the glass regions are indicated by green horizontal lines (Vertical streaking is an artifact attributable to the glass.) ND agglomerations are found on the outer edge of cells and, on occasion, in punctate spots inside the cells, verifying both that diamonds are able to enter the cells, and that they are concentrated in small areas, (d) Avalanche photodiode (APD) detection of fluorescence from NV NDs plus cellular auto-fluorescence showing that more sensitive detection provides similar detection of ND signals. Scale bar, 5 μιη. Figure 6 shows TEM confirmation of the feasibility of locating and imaging NDs distributed in a simulated biological slice of agarose gel. (a) Low-magnification image showing a slice with homogeneous appearance, and the field-of-view afforded by the copper support grid. Scale bar, 50 μιη. (b) Image of a ND embedded in the slice. White arrows indicate dark but non-crystalline regions of the specimen. The diagonal line is a knife mark from the ultramicrotome. Scale bar, 500 nm. (c) Magnified image of the single ND. The central horizontal and vertical lines are artifacts of the camera hardware. Scale, bar, 50 nm. (d) HRTEM image of the ND, demonstrating that lattice fringes in the crystalline can be resolved despite the significant background of the amorphous fixation resin. Scale bar, 0.5 nm.

Figure 7 shows an optical image of nanodiamond cluster inside of dehydrated cell after cryoelectron imaging of the same sample. ROIs are placed in the center of the diamond cluster (yellow) and the edge (blue). Line profiles are centered on the diamond cluster running horizontally (green) and vertically (purple). These line profiles are plotted in Figures 9 and 10. (ROI, region of interest).

Figure 8 shows intensity profile for line regions showing fluorescence (Exc. 561/543, Det. 630-800 nm) overlayed with transmitted light at 561 nm.

Figure 9 shows intensity profile 2D ROIs versus Z height for fluorescence. The locations of these two profiles are shown in Figure 7. (ROI, region of interest).

Figure 10 shows cryoelectron microscopy of the same diamond complex taken prior to optical measurements.

Figure 11 shows optical fluorescence image (zoom-in with respect to Fig. 7) of the same nanodiamond cluster as shown in Figure 10. Figures 10 and 11 are the sought correlated electron and optical images, respectively (the two images are shown on slightly different scales and orientations - the magnification level in Figure 10 is slightly higher than that of Figure 11).

Figure 12 shows a CAD drawing of generation #1 holder assembly. 4-way cell/reagent funnel and TEM grid holder used for optimizing seeding of cells onto TEM grids with uniform densities and enabling the TEM grids to be moved and manipulated without damaging them.

Figure 13 shows generations #1 (white plastic) and #2 (square with clear window and centered TEM grid) holders. The white plastic pieces are 3D printed from the CAD drawing shown in Figure 1. The square piece with a central square window and TEM grid mounted at its center is the generation #2 holder. Generation #2 holder contains a center object, which is a clear cover slip. It is wrapped with parafilm around the edges and supports a TEM grid. The razor blade is used to clear an open path to remove TEM grid without obstruction.

Figure 14 shows design for new TEM grid holder made from a standard coverslip, patterned with lithography and etched with hydrofluoric acid. It can include an embedded microwave antenna for electron spin resonance using NV ^" centers in nanodiamonds. Etching will be performed with lithography masking and formation of a similar design to the older, 3D-printed grid holder, which was opaque. Zoomed images show grid seated just over the edge of the cover slip and an etched space to secure the TEM grids, while still enabling access to the edge of the grids by tweezers. Depth of the grid holder wells and coverslip was chosen to be optimal for STED microscopy on the commercial Leica TSP SP5.

Figure 15 shows HEK 293 cells cultured on Quantifoil TEM grids, now with proper hole: support ratio to enable TEM and tomography. Image taken with light transmission microscope of an actively growing cell culture. Several images were stitched to provide wide area with high resolution.

Figure 16 shows 1 zoomed, 90° rotated view of the above grid shows many cells spread wide and with holes through the plastic support clearly visible. These regions contain only ~11 nm of conductive carbon film allowing imaging of cells with the absolute minimum material possible from the grid added. If the sample is not too thick, this ratio of hole to support spacing and hole support thickness are compatible with tomographic imaging and reconstruction capable of resolving protein structures.

Figure 17 shows a single cell identified in a corner of a well near the center of the grid to demonstrate correlated imaging of a desired cell chosen ahead of time. For simplicity all correlated images presented in subsequent Figures are of this cell with various zoom and color settings.

Figure 18 shows a thumb scale stitched mosaic reconstructed of HR-TEM image showing approximate overlap and image size.

Figure 19 shows a high resolution TEM (HR-TEM) stitched reconstruction of single cell for correlation from overlapping individual images, rotated and scaled for overlaying purposes. Figure 20 shows a single confocal section of red fluorescence, green fluorescence and phase contrast images taken as independent channels on a confocal light microscope with each channel merged to RGB and contrast-enhanced, then overlaid with high resolution TEM (HR-TEM) mosaic image. Grid holes and cellular features allow for accurate alignment.

Figure 21 shows an individual confocal slice of the green fluorescence channel (blue) and phase contrast (gray) images of single cell on TEM grid overlaid with high resolution TEM stitched image.

Figure 22 shows Optically Detected Magnetic Resonance (ODMR) of nanodiamonds generated by sweeping microwave frequencies from an antenna placed nearby the confocal microscope stage. ODMR response requires the presence of NV ^" centers in diamond. The optical response to microwaves, the ODMR signal, can be used uniquely both to distinguish a nanodiamonds from other materials and to distinguish distinct nanodiamonds from each other.

Figure 23 shows an image of room temperature TEM with resin embedded and heavy metal staining of HELA cells showing clearly identifiable nanodiamonds in small clusters. A typically observed pattern of targeting is shown, with Nup91 antibody causing the nanodiamonds to target at the outer edge of the pore on the cytoplasmic side and closer to the membrane on the nuclear side. Geometry of nuclear pore matches that presented in the literature. This also indicates delivery of nanodiamonds to the nucleus. The presence of both labeling locations in one screen shot attests to the specificity of the labeling to cellular regions that Nup91 is known to localize to. (i.e., both the cellular side and the nuclear side of the nuclear pore).

Figure 24 shows an image of nanodiamond with clear Fresnel fringing observed under bright field, which enables unambiguous identification of the diamond by even untrained observers, which is uncommon for staining in biological TEM. The location of the nanodiamond is approximately ¾ of the way down along the vertical direction, and half-way horizontally (i.e. near the center along the left-right direction).

Figure 25 shows a resin embedded cell with several nanodiamonds successfully labeling nuclear pores showing the ability of the antibody conjugate to deliver nanodiamonds into the nucleus. (The nanodiamonds appear smaller here than in Figure 24, but can still be seen, along with the Fresnel fringing.) Figure 26 shows nanodiamonds aggregated in endosomes (likely lysosomes), indicating a possible macro aggregation of lysosomes containing nanodiamonds to a common cellular region, possibly a disposal or storage site. The sample was prepared by growing HEK 293 cells directly on Quantifoil TEM grids, drop frozen in liquid ethane to preserve as vitreous ice and the image was taken using an FEI TF20 cryo electron microscope in bright field mode at high magnification. Scale bar, 200 nm.

Figure 27 shows cellular structures revealed by cryo electron microscopy in a natural live cell state. Vitreous ice represents a frozen snapshot of a living state and this image shows that the native densities of proteins provide enough contrast to visualize macro molecular assemblies in living cells.

Figure 28 shows a bioluminescent nanodiamond, which is coupled to luciferase.

Figure 29 shows excitation transfer efficiency for different types of luciferases coupled to nanodiamonds.

Detailed Description

There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could allow targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here, this disclosure demonstrates a sub-cellular multi-modal imaging methodology that allows localization of inert particulate probes, corresponding to nanodiamonds (NDs) having fluorescent nitrogen- vacancy (NV ) centers or other fluorescent sites. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. NDs targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to allow "zooming-in" to regions of interest for detailed structural investigations. Optical microscopies reveal NDs for in vitro tracking or uptake-confirmation. The approach is general, works down to the single ND level, and can leverage the unique capabilities of NDs, such as biocompatibility, sensitive magnetometry, and gene, protein and drug delivery.

This disclosure describes a system, method, and device that allows one or more of the following by an approach for a delivery vehicle that is also, simultaneously, a biomarker resolvable by both optical and electron microscopies. Kits including containers and compositions including NDs are also encompassed by this disclosure. Embodiments employ NDs containing strongly fluorescing nitrogen vacancy (NV ) color centers (or any other color centers which are sufficiently fluorescent to be detected in the context of a study) that allow a simultaneous combination of all (or a subset) of the following goals: (1) Biological delivery of drugs, genes, or diagnostic agents into the cytoplasm of living cells (e.g., in vivo), or onto surface-exposed membranes or penetrating into the cytoplasm in fixed/frozen/thawed slices; (2) Targeted delivery of the agents of (1) to specific cellular or sub-cellular membranes (e.g., an organelle the nucleus, or a foreign particle, agent, virus, or organism); (3) Provision of typical and super-resolution optical microscope imaging in vivo (or in fixed, frozen, or sliced samples), and characterization of samples (such as delivery success rates or diagnostic confirmation of presence of agents) by using the strong fluorescence of NV ^' NDs (which are color centers buried within the NDs, and are thus non-cytotoxic; other color centers in diamond can also be used, if sufficiently fluorescent); and (4) Provision of TEM imaging either in a slice or even in vivo with the use of an environmental TEM. This is allowed by the possibility to image the ND delivery vehicles/markers separately from cellular superstructure using electron-scattering effects for contrast (e.g., diffraction, high-resolution TEM (HRTEM), high angle annular dark-field scanning TEM (HAADF-STEM), STEM, bright- field diffraction contrast through the use of an exclusive objective aperture to yield contrast, or other techniques present or future). Any or all of these are allowed by the use of nanoparticles of a single type (NV ^~ bearing NDs or NDs containing other suitable color centers). Any or all of these are improvements over other approaches by their (a) characteristic non-cytotoxicity; (b) their biological inertness; (c) their non-bleaching character; and (d) their non-blinking fluorescence, which are problems associated with other approaches to both intracellular agent delivery and particulate marking of sub-cellular structures. The approach described herein can meet all of these capabilities in a single vehicle/nanoprobe. It is also noted that the NV ^" color center is strongly fluorescent, which is a desirable feature when imaging cells or tissue that are already autofluorescent: it allows localization of the ND above the existing background fluorescence.

Applications of this disclosure include gene therapy, protein therapy, drug delivery, targeted cancer therapies, and a host of medical diagnostic techniques (that can confirm, rule- out, or locate foreign agents, viruses, biological organisms, cancer signatures, and so on), whether existing or to be developed in the future. Further applications of the disclosure are the use of the NDs as alignment fiducials for TEM imaging or TEM tomography (tilt-series of images), as a post-fixation stain as an approach to targeted placement within cells or biological structures, for use of the NDs as temperature probes, MPJ contrast agents, cellular division tracking fiducials, and so forth. The approach is appropriate for staining/marking live cells, cultures, fixed cell samples, as well as slices (fixed, frozen, or thawed), or cryo- fractured samples. Another application is the correlation of optical and electron microscopy images; this is made possible because the fluorescent NDs can be easily detected by both modalities simultaneously. This allows the user to combine the strengths of both imaging modalities when studying biological features. Another application is to allow tracking of biological and sub-cellular processes by either or both optical microscopies or TEM. The approach allows iterative imaging between the two techniques.

Advantages and features of the NV ^' ND probes/delivery vehicles include one or more of the following:

- NDs conjugable delivery vehicle - enhancing both drug/therapy/diagnostic delivery and imaging to allow confirmation of delivery to a specific biological site (e.g., a subcellular organelle).

- NDs can be conjugated with therapeutic agent (drug, gene therapy, protein therapy), or diagnostic agent (diagnostic chemicals).

- NDs can simultaneously be conjugated with antibody-specific antigens, to target specific subcellular structures or the outer membranes of specific cell wall to "mark" specific structures, and can also be used as delivery vehicles for drugs, genes, or diagnostic agents.

- The ND probes can be transfected into live cells, allowing in vivo optical, and even TEM studies with the use of an environmental TEM instrument and/or holder (generally comprising ultra-thin membranes sandwiching a living biological sample and a differential- pumping aperture(s) in the TEM, although other embodiments of such exist). This approach is preferably used by applying low-dose electron imaging techniques (known in the art), in order to avoid excessive cell damage.

- The approaches of environmental TEM holders (as above) allow imaging live cells in an iterative fashion, moving back-and-forth between optical and electron methods. This allows numerous types of tracking, cell growth, cell division, and other studies, as well as diagnostics.

- On frozen or fixed samples, HRTEM of sub-cellular structures is made possible, and damage to the sample by the electron beam is reduced significantly because the ND landmarks allow rapid alignment and calibration of the TEM. - With environmental TEM, the method described herein also allows rapid alignment of the TEM field of view, and minimization of the electron dose to the sample.

- NDs can be conjugated with both a cell membrane-specific antibody and with a drug or diagnostic that it is desirable to deliver for therapy or diagnoses.

- The NDs can be transfected into live cells. This can be done without the use of detergents for trans fection (which kills cells). PPI dendrimers can be used, but other reagents, existing and future, may be used. Direct transfection into the cell using microinjection techniques is also possible.

- The ND nano probes are visible from both optical and electron microscopy images.

- The ND nano probes are biocompatible (non-cytotoxic)

-The ND nanoprobes' optical fluorescence signal does not degrade to any significant degree within the duration of an experiment (or during the lifetime of a sample). Its signature in the electron microscope also does not degrade significantly over time because of the superior robustness of the diamond material compared to other materials.

- The signal is significantly more temporally stable than most fluorescent molecules, allowing tracking of single particles continuously in time without blinking (a problem with QDs), preserving the ability to track a ND particle-marker in vivo.

- The ND probes are chemically stable and inert. They do not bleach.

- The ND probes allow optical and electron imaging with lower radiation dose to the sample (low radiation dose is an important aspect, as radiation dose can damage biological samples under study).

- The ND probes allow detection from lower magnification in both imaging modalities because of high quantum efficiency as a fluorophore for optical detection, and high contrast in electron microscopy due to its difference in electron-scattering angular distribution, which allows numerous TEM techniques based preferably on techniques which use a subset of scattered electrons for detection. Some, but not all, of these techniques involve the use of a restrictive objective aperture to increase image (real-space) contrast, recording diffraction patterns for confirmation of ND crystallinity at a specific site, scanning TEM (STEM) techniques which make use of the above-mentioned differences in electron scattering such as high-angle annular diffraction STEM (HAADF-STEM), conical diffraction STEM, and others known in the art or to be developed in the future. - Other preferred TEM modalities rely upon chemical-sensitive techniques such as STEM electron energy loss spectroscopy (STEM-EELS) or "EELS mapping," in which case the mechanism of contrast would not be electron-scattering based, but would be based upon measuring energy lost in inelastically scattered electrons in a suitable TEM, or on the measurement of x-ray resulting from the same physical process. In either case, nitrogen or oxygen would be mapped, so that "darker" areas could be inferred in images as NDs, because NDs are composed of carbon.

- A further embodiment of TEM imaging is to use Fresnel fringes that form on the edges of NDs in a sample as the image is defocused. This approach is used in an environmental TEM setting, where cell-stains such as osmium tetroxide and lead citrate are not used, because those also introduce contrast that could confound image interpretation. For this TEM modality, it is also desired to have a thin slice of biological material to reduce the amorphous background contrast that arises from the biological sample. Slices can be less than 200 μιη in thickness, or less than 80 μιη in thickness, or less than 40 μιη in thickness. Sample infusion with NDs for this modality can be done as decoration of a biological slice (thawed, embedded, or fixed). It could be employed on an in vivo ND-infused biological sample, but the similar size of the diamond knife and the ND markers may complicate accurate image interpretation.

- The probes offer features that aid in identification in optical microscopy using the coincidence of two uncommon signatures under fluorescence lifetime imaging (FLIM); a short signal approaching the instrument response time (e.g., 200-400 picoseconds and a long fluorescence lifetime often well in excess of 17 nanoseconds in comparison to common cellular fluorescence lifetimes of 1-5 nanoseconds). The identity of the nano probes can also be identified by optically detected magnetic resonance (ODMR), with the addition of a microwave field to manipulate the magnetic sublevels of the paramagnetic electrons in the color center. In one incarnation, the microwave field can be used to modulate the fluorescence intensity of the NDs. Other fluorescence-based optical and ODMR imaging techniques, known in the art or developed in the future, can also be used.

- The ND nano probe can be used in live cells or inert material to measure local temperature and magnetic fields to high accuracy and precision, and possibly also in a successive step have their locations imaged or confirmed by TEM. The advantage of performing TEM imaging would be the additional benefit of resolving subcellular structures in proximity of the NDs, enabling a more accurate interpretation of the results. - The ND nano probe can be used in live cells or inert material to measure the local concentrations of paramagnetic centers or molecules (such as free radicals, or transition metal atoms in molecules).

- The ND nano probes can be covalently or non-covalently bound to any biological or organic molecule or combination of molecules by established chemistry, and it has been demonstrated that this attachment can be used to target the nano probe to locations determined by the attached molecule.

- This ND nano probe can be used to deliver a functional protein directly into the cytoplasm of a cell demonstrated by the covalent attachment of an active antibody, such as a suitably soluble antibody.

- The identity of the ND nano probe can be confirmed in both modalities: for TEM, through the use of electron-sample interactions which are characteristic of crystalline particles, physical inclusions in a sample, or its signature (nearly) pure carbon composition, all of which are characteristic of NDs in a sample, and are absent from native cellular structures; for optical microscopy, by the unique fluorescence signature of the color centers in diamond (fluorescence spectra, lifetime and ODMR response).

- In some embodiments nanodiamonds are less than or no greater than 200 μιη in size, less than or no greater than 60 μιη in size, or less than or no greater than 30 μιη in size. The smaller the nanodiamond, the greater its mobility and ability to transport across membranes and tissue. However, NDs that are too small (e.g., less than 10 μιη in size) may be difficult to locate in the TEM images in some embodiments. A good size range to work with is 30-100 μιη in some embodiments. The NDs should be sufficiently crystalline, so that electrons can be diffracted.

- The ability to be identified in low number on the order of 1 per tens of cells using both methodologies within the standard times used for electron and optical imaging.

- Functionalized and targeted probes are able to escape endosomes (transfect), and diffuse into the cellular cytoplasm and attach to their target of interest. This is a desirable aspect of using any particulate nanoprobe or marker. NDs (and other marker particles) can be trapped in endosomes or other foreign-body trapping responses that cells can exhibit. A ND nano probe that is trapped in an endosome or similar structure is effectively rendered biologically inert because it is not free in the cytoplasm. The approach of this disclosure uses propylene polyimide (PPI) dendrimers as a transfection agent, which allows transfection without damaging or killing cells. Other transfection agents known or future may be used.

- In another embodiment, detergents, which are typically used to transfect indicators, albeit into dead cells, can also be employed to transfect the NDs.

- The ND nano probes can be used for polarization transfer of an optically imparted spin polarization to neighboring nuclei or electrons on separate biological and organic molecules in live cells.

- The ND nano probes can be used through several cycles of freezing and thawing of tissue for repeated cycles of optical and electron imaging with a sample that can tolerate the process (e.g., North American wood frog which freezes annually) without any significant signal degradation in either modality.

- The ND nano probe emits a strong optical signal in the near infra-red (for NV ^" NDs) which penetrates tissue deeper than shorter wavelengths, and the probes can be excited by multi-photon absorption which allows excitation of this fluorescence deeper than the emission wavelength can penetrate.

- The methods used to image with this probe are consistent with both tissue cultured cell monolayers and tissue slices from living tissue obtained from animals or human patients.

- The ND nano probes can be modified by electron irradiation in a standard electron microscope to assist in the formation or directly in the destruction of NV ^" centers, altering the probes' fluorescent properties and allowing unique identifiers or bar codes.

- The method can be used solely for electron microscopy. If optical microscopy is not used, then the presence of color centers in the NDs is not required.

As noted above, NDs can be conjugated with antibodies or other specific binding agents. An antibody can refer to an immunoglobulin molecule or immunologically active portion thereof, namely, an antigen-binding portion. Examples of antibodies that can be used in the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, recombinant antibodies, an affinity maturated antibody, single chain antibodies, single domain antibodies, F(ab) fragments, F(ab') fragments, disulfide-linked Fvs, antiidiotypic antibodies, and functionally active epitope-binding fragments of any of the above. A specific binding agent can refer to a member of a specific binding pair. That is, two different molecules where one of the molecules, through chemical or physical means, specifically binds to the second molecule. In addition to antigen and antibody specific binding pairs, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, nucleic acid duplexes, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog or a mutated enzyme in one or more amino acid positions, or a mutated/altered (e.g., non-complementary) nucleotide sequence. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal and complexes thereof, including those formed by recombinant DNA molecules.

Studies on gene, cancer, and drug therapies could benefit by the use of a single nanoparticle (NV ^~ NDs) for both delivery to a specific biological site, and also functions as a biomarker for subsequent microscopies to confirm uptake, or to measure success rates of targeting (e.g., treatment efficacy). Because electron microscopy images can involve fixing the cells (for epoxy resin preparation), or freezing the cells (vitreous ice technique), or encapsulating the cells into an environmental chamber, it can be desirable to first perform characterization in the live state, such as optical fluorescence imaging. Electron microscopy is then performed on the samples. If a live cell imaging study is desired, the samples can be shuttled back and forth between electron and optical microscopes. To this end, a suitable substrate/grid can be used. Vitreous ice techniques allow the experimentalist to thaw the cells back to life for further characterizations. Repeated cycles of optical/electron imaging are possible using the vitreous ice technique.

The method for use of the nanoprobes can include all, or a subset, of the following steps:

- Addition of fluorescent centers into nanodiamond probes (as an example, negatively charged nitrogen- vacancy centers can be used, as they offer strong fluorescence).

- Surface functionalization of the NDs (for targeting and transfection into the cell) via polypropylene imide (PPI) dendrimers or other suitable targeting and transfection agents.

- Surface functionalization with an antibody-specific antigen to allow targeting to a specific cellular or subcellular bilipid membrane or transmembrane complex. When placed inside an electron microscope, NDs can be selectively irradiated (with various electron doses). This irradiation can be used to selectively create or destroy NV ^" centers, or biological tissue. Such selective irradiation could be useful as a way to create spatial signatures (landmarks or fiducials) for positioning or for sample analysis.

- Transfection of cells with NDs using maltotriose conjugated PPI dendrimers or similar technique.

- Samples for electron microscopy can be embedded with epoxy resin, or rapidly frozen using cryogenic techniques (vitreous ice).

- Alternatively, NDs can be used to stain an embedded or cryo-slice, or a vitreous ice fractured sample.

- Optical and electron imaging of the samples on a suitable grid or film.

- Electron imaging uses diffraction-mode scan to rapidly scan for NDs throughout the sample and discriminate diamonds from non-diamond structures (which can appear indistinguishable in real-space mode images).

- Following identification of NDs in diffraction mode, real-space imaging is used to "zoom-in" into the region of interest in a cell and obtained detailed nanoscale-resolution images of the subcellular structures of interest. Alternatively, NDs can be localized in real- space mode using Fresnel fringing. The localization of NDs from diffraction features could be automated on a computer to accelerate the process and further minimize electron dose to the sample.

- The selective destruction of color centers in the nanodiamond probe (for example, as a way to watermark or fingerprint a subset of nanodiamonds within a constellation).

- Optical images are acquired using the fluorescence signals originating from color centers in diamonds.

- Live-culture or fixed-slice imaging of biological samples by optical microscopy techniques.

- Live-culture or fixed-slice imaging of biological samples by TEM.

- Multi-modal imaging by optical and electron techniques, even in an iterative or "back-and-forth" manner.

- Ability to perform correlated imaging on commercially available electron microscopy sample grids, or suitably modified or annotated grids for purposes of alignment and correlation. - Ability to find the identical, same nanoparticles in a single sample preparation on optical and electron images (with both modalities).

- Method for extracting NDs from biological materials/samples for further analysis.

The feasibility of the technique is demonstrated to locate targeted NDs inside a cell using electron microscopy. Optical images are obtained of the fluorescent NDs transfected into cells. Correlated optical and electron microscopy images are demonstrated. The sample preparation method used is epoxy resin preparation, as well as vitreous ice techniques. Clear detection of the nanoprobes in vitreous ice is demonstrated using low magnification diffraction based detection, verifying their identity with diffraction patterns and Fresnel fringes from the diamond crystal structure in vitreous ice, and verifying their ability to escape endosomes. Site specific targeting using vitreous ice techniques and the use of environmental electron microscopy also can be demonstrated.

ADDITIONAL EMBODIMENTS

Described here are system, method and device based on sub-cellular multi-modal imaging methodology that allow localization of inert particulate probes, corresponding to nanodiamonds (NDs) having fluorescent nitrogen-vacancy (NV) centers or other fluorescent sites. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. The approach works down to the single ND level, and can leverage the unique capabilities of NDs, such as biocompatibility, sensitive magnetometry, thermometry, gene, protein and drug delivery.

Nanoparticles have emerged in recent years as a promising approach to particulate labeling probes for multimodal imaging, and also for targeted drug, protein or gene delivery. In general, little is known about the immediate environment of targeted nanoparticles, due to a lack of suitable visualization protocols. The development of super-resolution techniques has improved spatial resolution. However, these techniques are restricted to a subset of cellular processes. Transmission electron microscopy (TEM) offers higher spatial resolution. Yet, it lacks reliable multimodal markers to align the field-of-view with a desired subcellular region. TEM also lacks diversity of available stains and markers compared to other microscopies.

Gold can be cytotoxic by itself, and frequently becomes isolated in lysosomes in live cells. Colloidal quantum dot (QD) biolabels are cytotoxic, and suffer from "blinking" problems and are not suitable for human use. Several candidate markers for multimodal imaging have been pursued, but none has yet been found to be practical and universally applicable. Delivered DNA/R A/protein degrade quickly in a cell environment in free form or when attached to a delivery reagent or vehicle. Further, detection of conjugate biodistribution is a limiting factor in FDA trials. In addition, long-term accumulation of conjugates is difficult to detect if probe degrade over time.

To solve these problems, described herein are composition and method that allow localization of inert particulate probes, corresponding to nanodiamonds (NDs) having fluorescent nitrogen-vacancy (NV ) centers or other fluorescent sites. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Optical detection can be enhanced by the combination of NDs with existing optical labels.

NDs targeted to a cellular structure of interest (such as the nuclear pore complex) are rapidly localized in electron-microscopy diffraction mode to allow "zooming-in" to regions of interest for detailed structural investigations. NDs bridge the gap between biomarkers available for optical imaging and high resolution electron microscopy methods.

NDs possess characteristic non-cytotoxicity and biological inertness. They overcome the problems associated with other approaches for intracellular agent delivery and particulate marking of sub-cellular structures with their non-bleaching character and non-blinking fluorescence. Further, NDs can be assayed with simpler techniques (i.e. needle biopsy) because of their optical fluorescence (e.g. NV ^" center) for investigation of biodistribution. In addition, NDs rarely degrade mechanically or optically, enabling assays to be done over an entire lifetime.

In some embodiments, a biological material is brought in contact with a composition comprising NDs. NDs nano-probes can be conjugated with therapeutic agent (drug, protein, gene therapy), or diagnostic agent (diagnostic chemicals), or any biological or organic molecule or combination of molecules by established chemistry. Once conjugated, the NDs can be utilized (a) to target cells, specific sub-cellular structures, specific membranes, viruses or pathogens to "mark" specific structures; (b) to extend the active lifetime of delivered molecules/reagents; (c) as delivery vehicles for drugs, genes, proteins or diagnostic agents; and (d) to transfect live cells, allowing in-vivo optical and TEM studies with the use of an environmental TEM instrument and/or holder. The nanodiamonds can have non-cytotoxic character, ability to enhance the in vivo lifetime of delivered molecules, biological inertness, ability to transfect into the cytoplasm without trapping into endosomes (e.g., through the use of dendrimers), independence from a protective layer, high water solubility, non-bleaching character, non-denaturing character, and /or non-blinking fluorescence.

In addition, the nanodiamonds can function as a delivery vehicle and/or a biomarker in targeted delivery of agents to extracellular and intracellular structures (such as those within the cytoplasm, extracellular matrix or within the nucleus) and/or surface-exposed membranes.

This approach described herein exploits the unique physical, chemical, and biological properties of nanodiamonds containing fluorescent nitrogen vacancy centers to enable a single marker or delivery vehicle to be observed in a live cell/tissue/animal/or human environment (fluorescent imaging), using electron microscopy for visualization and localization of the same individual or group of markers. A significant advantage compared to other available materials in the area of correlated imaging derives from the ability of this marker to enable unrestricted use of the full set of technologies applicable to fluorescent imaging, including but not limited to cameras, STED which can be non-bleaching and non- blinking, PALM/STORM/GSD which can be photo-controllable, lifetime (17ns lifetime, vs 3 -5ns from cells) microscopy, confocal microscopy, phase contrast microscopy, and transmission microscopy, while imposing no restrictions on electron microscopy methods and supplying several unique advantages to both methods.

The approach described herein uniquely enables live cell fluorescent imaging of single particles (virus, protein, gene delivery vehicle, etc.) followed by electron microscopy under conditions compatible with cryo EM tomography which enables visualization of proteins unstained and in their native state with their native binding partners. The inertness of nanodiamonds contributes to this directly by providing the best case possible for preserving the native state due to the chemically inert nature of the nanodiamonds. Nanodiamonds are carbon based, making them similar to organic molecules, but their crystalline causes them to deflect electrons in a significantly distinct manner from noncrystalline carbon that predominates in biological materials and cells. The crystalline nature of nanodiamonds allows diffraction patterns to be obtained on the electron microscope, which can be used to identify diamond unambiguously, even from other crystalline materials. These physical distinctions enable discrimination between nanodiamonds and most biological or cellular materials at a signal to noise ratio that is exceedingly high in both modalities compared to other labels that are available. These physical properties enable the identification of nanodiamonds across at least 7 orders of magnitude in non-crystalline organic material. This is significant for structure determination because electron imaging is destructive at the structural level and energy used to locate a region can significantly damage the sample, reducing the total number of images that can be taken before degradation. The use of low magnification localization reduces the energy intensity preventing significant damage prior to high magnification imaging. The ability to detect nanodiamonds with sensitivity capable of identifying single nanodiamonds enables experiments where 1-10 total nanodiamonds per cell can be used and reliably found, enabling a minimally perturbed cell state reflective of the native environment and naturally occurring binding partners for any attached moiety.

NV centers can be used to do magnetic measurements, temperature measurements and electric field measurements with nanoscale resolution using fluorescence. The approach described herein extends this ability to include structural information about the environment being scanned which can be correlated with the magnetic information obtained from the same sample kept in an identical state. NV centers can also be used for temperature measurement to milliKelvin precision using fluorescence detection and a microwave field, and it is uniquely NV ^" centers that can allow the direct correlation of structure with temperature, electric fields and magnetic measurements in the same sample.

Surface chemistry of nanodiamonds enables rapid, covalent or affinity based attachment of diverse molecules or moieties. Carboxylation improves solubility and the ability to control the carboxylation allows control of the number of conjugated molecules and allows for sequential addition of conjugated partners using protection groups with well- established chemistry. Again the use of nanodiamonds for correlated imaging includes the benefits of an diverse and robust set of attachment methods, all of which further the power of the imaging methodology by allowing its application to a broader set of applications.

The approach described herein offers a major translational advantage because of its suitability for analysis in basic science, applied sciences, medical research, preclinical studies, clinical trials, post clinical analysis and even post mortem analysis utilizing the same markers and/or preparations. The approach described herein imposes almost no additional restrictions at any stage compared to other markers and offers unique advantages or comparable performance for almost all of the important analysis techniques used in these areas. The use of NV ^" containing nanodiamonds in correlated imaging enables determination of unknown protein structures in a live cell state captured with minimal perturbation from the ND marker or with zero effect if nearby repetitive features can be used at a fixed distance from the marker. With the low detection threshold of nanodiamonds using TEM, it is possible to target a small number of individual proteins, possible introduced as foreign or native.

Diamonds and the NV ^" defect are also resistant to almost all chemical treatments, including harsh preparation methods commonly used in electron microscopy, temperatures up to 500 °C, and no alteration in time. Spin coherence measurements can even be used to uniquely identify individual diamonds for months or years. They can also be easily purified by exploiting the stability of diamond and by centrifugation, which exploits the density difference between crystalline materials and organic materials.

Centrifugation enables the use of diamonds to act as a trap for targeted or untargeted proteins and can then be pelleted and analyzed using TEM outside of cellular contexts. Using this strategy fluorescently labeled diamonds can be produced with controllable numbers of capture and target proteins/complexes, which are then able to be analyzed by fluorescence for quantification and by TEM for characterization, both of which use each microscopy in its strongest context, wide area and high resolution respectively. The unique capabilities of the NV ^" defect enables magnetic field, electric field, and temperature sensing enable the NV ^" center in diamond to sense the immediate environment surrounding it. This ability allows detection of changes in surface conjugated moieties, which can be signal enhanced (such as gadolinium), N15 doped protein, etc., by fluorescence measurements at the single molecule level. This could be used to measure cleavage of standardized proteins as a standard blood workup for healthy patients, in which trace nanodiamonds are administered to reach the bloodstream and after collection potentially from any body fluid, excretion or tissue, or in vivo measurements in the skin or surface regions with high blood flow could be used to assess healthy body functioning, metabolic shifts, cancer, pathogens, etc. Mechanisms of cleavage, substrate modification or protein adsorption serve as examples of the diverse in vivo blood/body assays that could be read out optically using the NV ^" defect present to determine the immediate environment around the nanodiamond with state of the art techniques, especially in low concentrations or without an enhanced signal in a constrained setting. This approach enables the determination of the surrounding context in a true to life stable form, which allows assessment of the correlation between the detected optical signal of interest and the exact structural state of the immediate surrounding environment around the nanodiamond on a particle by particle basis. This approach enables unique advantages in removing ambiguity (suitable for a gold standard) and the ease of analysis made possible by the nanodiamond being able to be used as a correlated marker. There are major limitations and/or restrictions on other techniques or materials for this technique and no material will likely facilitate so many features so as to allow the technology to move out of research applications and directly in patient care. In the context of evaluating complex or novel signals, utilizing reliable and distinct changes that are correlated to strictly controlled sample changes can be used to generate distinctive but reliable signal "fingerprints" that report on the underlying states of interest generated by controlled sample preparations. The ability of TEM to resolve targets to the atomic scale, produces such high information "fingerprints" that the information required to unambiguously resolve questions of interest about the environment immediately surrounding the nanodiamond, especially for biological problems (aggregation state, binding partners, etc.). Once rapidly standardized by TEM/fluorescence correlation a standard "fingerprint" set could be determined for optical based sensing and an all optical in vivo assay could be well characterized and assessed on a gold standard system without any alteration to the medical device or vehicle (nanodiamond).

The approach described herein allows in vivo assessment of the conjugation status of the delivery vehicle and its deliverable payload or permanent conjugate using optical methods, but with nanoscale sensitivity characteristic of NV ^" centers.

Electron microscopy can be used to modify NV ^" content in nanodiamonds by producing defects, allowing tagging or enhanced discrimination of individual diamonds. Defects can enhance or reduce the number of NV ^" centers depending on the existing material.

Nanodiamonds have been tested more thoroughly than most nanoparticles and have a demonstrated track record of non-toxic use in humans, mice, and extensively in tissues and cell lines. To date, few experiments have demonstrated any significant toxicity associated with nanodiamonds, especially in physiologically relevant dose ranges. The inherent tolerance of the body for the presence of nanodiamonds limits the extent to which they activate inflammation, antibody responses, sequestration and complementation factors.

The approach described herein represents a threshold to enable use of several cutting edge microscopy technologies in combination with long term in vivo use. It will enable long term monitoring of biodistribution of the delivery vehicle via fluorescence or needle biopsy in living patients. The nanodiamonds and the NV ^" fluorophore can survive post mortem recovery for biodistribution by the harshest solvents, acids, bases and other chemical treatments.

The approach described herein is advantageous in terms of biocompatibility. It enables statistical assessment of on-off times and targeting for agents on ND delivery vehicles in live tissue. It also enables assessment of tissue, cellular, subcellular, and the immediate environment of the nanodiamond and includes the ability to assess whether vehicle and drug are still co-localized. The approach described herein is also advantageous in terms of automated quantitation of biodistribution by electron microscopy. It uniquely enables accurate large area sampling of tissues at any stage. Combined with automated detection it can be applicable to high throughput processes as well.

In some embodiments, nanodiamonds are composed principally of carbon, like organic molecules, except that it is crystalline. This distinction creates significant changes in the optical properties of the material. Crystals diffract light in uniquely identifiable ways meaning that nanodiamonds can be molecularly identified, and possibly individually distinguished by diffraction spot projections. Crystal quality affects diffraction, and nanodiamonds have an ideal strength and stability to ensure that small crystals are well crystallized. TEM is the primary means of determining diffraction projections in small samples, which provides an unambiguous method to prove that the object in question is diamond and not another crystal (such as ice, aggregated proteins, etc). This means that with a filter that specifically removes diffracted light or electrons the nanodiamonds appear uniquely black, standing out clearly with high signal to noise ratio from all biomaterials. The second effect of crystalline material is Fresnel Fringing, in which crystal structures appear with a glowing ring when defocused, and artificial darkness when overfocused. This effect is also visible in light microscopy, and while not emphasized here, also contributes to the uniquely distinguishing features under optical microscopy along with unique lifetime, emission frequency, magnetic resonance and optical stability. By imaging a biological sample over focused, exactly focused, and defocused, it is able to rapidly distinguish crystalline features from non-crystalline, which removes the vast majority of background items. Third, under a modulated eucentric focus diamonds appear to twinkle, allowing them to be identified in a fast dynamic way without image processing. ADDITIONAL APPLICATIONS

The approach described herein can be used for cancer detection through NV-diamond circulation followed by structural imaging. Circulating diamonds can adsorb or specifically bind protein. Fluorescent imaging can be used to analyze co-localization

The approach described herein can also be used to assess drug/vehicle uptake or substrate cleavage by macrophages from cancer patients or healthy subjects using whole blood. It is able to distinguish routes of entry, whether internalization is able to harm blood cells, if they are stored in lysosomes or broken down rapidly.

The approach described herein can also be used for personal diagnosis. The nanodiamonds composition can be given to patients where the nanodiamond efflux can be collected and returned to the lab for personal diagnostics.

The approach described herein can also be used for viral detection, localization, and identification by structural analysis during a viral outbreak.

The approach described herein can also be used for magnetic fingerprinting coupled with structural information for rapid viral identification in an outbreak.

The approach described herein can also be used for melanoma FDA trial to assess subcellular localization and biodistribution via bio luminescent imaging in surface tissues.

The approach described herein can also be used for mouse drug/gene/protein study to assess long term colocalization of therapeutic agents and ND delivery vehicle during xenograft challenge.

WORKING EXAMPLES Example #1

This example describes the use of bioconjugated NDs as markers for locating target cell structures. Locating ND landmarks by regular (i.e., real-space imaging mode) TEM can be problematic due to the strong background signal originating from the amorphous fixation medium. For example, several features in such TEM images can resemble nanodiamonds in real-space images, without being nanodiamonds. To this end, the method described herein enables the user to unambiguously identify the NDs over other features. With TEM, this is done by examination of the electron diffraction patterns from NDs, which exhibit a unique signature not present in cells and biological media. (To this end, lattice-resolution TEM or electron diffraction is desired.)

It is noted that a common problem with NDs is that they tend to agglomerate, reducing the bio-availability of conjugants, which tend to remain outside of cells, adhere to the outer cell membrane, and when taken up generally remain trapped in endosomes, all of which render them biologically inert. To overcome this problem, the surfaces of NDs used are conjugated, functionalized and targeted.

The NDs are implanted with NV ^" centers and targeted using a nuclear membrane- specific localizer protocol where single NDs are transfected into the cytoplasm of live HeLa cells. After embedding and slicing, the cells are imaged by fluorescence lifetime imaging (FLIM), confocal microscopy and by TEM — bright-field, lattice fringe imaging, and electron diffraction— to unambiguously differentiate NDs from similar-looking features. The bright fluorescence of NV ^" centers in NDs yields excellent cellular labels. Confocal fluorescence microscopy confirms that control (untransfected) cells incubated with an excess of NDs primarily form aggregates of NDs outside cells, with occasional NDs internalized and retained near cell. It is noted that different color centers (different than NV ^" centers) can be used in NDs, provided they yield sufficient fluorescence. Often, the brightest color centers in NDs are obtained by implantation and/or irradiation.

Results: For lower ND-concentration imaging, FLIM is used. The DAPI stain serves as a positive control for lifetime imaging (Fig. la), where the nuclei are clearly resolved from the cytoplasm and from areas outside the cell by differential fluorescence lifetimes. The secondary peak in fluorescence is consistent with DAPI having a stronger signal and a clear peak at the expected lifetime of 2.2 ns. This peak can also be resolved into components below 1 ns and above 3 ns, representing bound and unbound DAPI, consistent with the expected emission of DAPI from two and three-photon absorption processes which both occur at 910 nm. This signal dominates. Cellular auto-fluorescence, which had the strongest measured lifetimes between 1 and 3 ns, is consistent with literature values (Fig. lb). Together these signals serve as strong positive controls for the accurate measurement of fluorescence lifetimes. NDs exhibit an short optical emission lifetime, on the order of 200 ps (Fig. lc). The histograms of lifetimes weighted by pixel intensity show two peaks, one that corresponds to the short emission lifetime of the NDs (about 250 ps), and a second that corresponds to the cellular auto-fluorescence and/or DAPI signals. The NDs in cells show the expected distribution, appearing as punctate spots with exclusion from the nucleus (Fig. lc).

Conjugation and transfection were used to deliver NDs into living cells (in vitro), to help them escape from endosomes, and to be released into the cytoplasm. Fluorescence lifetime imaging of untransfected NDs (Figs. 2a, b) confirms the fluorescence imaging results above. Lifetime imaging of cells transfected with NDs conjugated to anti-actin antibodies using PPI dendrimers (Fig. 2c) confirms successful transfection by the broad distribution of NDs throughout the cytoplasm, which is distinct from distributions of nanodiamonds when confined within endosomes or targeted to specific membrane-bound structures. The exclusion of signal from the nucleus provides additional evidence that the NDs are responsible for the short lifetime component. Conjugation alone does not produce a broad cellular distribution, but PPI dendrimers significantly enhance the release of NDs into the cytoplasm (Fig. 2d), promoting conjugation to membrane antibodies.

Imaging of individual NDs in a biological TEM specimen presents several challenges. Because the ultimate goals are to allow multiple iterations of imaging, and ultimately to perform correlated imaging, cell cultures were first embedded in epoxy resin before slicing by ultramicrotome, with slices ranging from 70 to 250 nm in thickness. The mounting resin creates a significant amorphous background that confounds TEM image interpretation, particularly for objects under 100 nm in size. A further complication is that NDs and the mounting resin have essentially identical electron densities. Together, these hinder the use of commonly used methods for achieving image contrast in a nano-inclusion. That is, underfocusing the image to achieve Fresnel contrast at edges is not practical (in this particular sample preparation), as contrast derealization in the mounting medium causes a strongly modulated background that can obscure such detail, even at limited defocus. Second, there is no appreciable mass-thickness contrast between NDs (carbon) and the mounting medium (carbon, oxygen, nitrogen). There is also the complicating factor that a stained sample (osmium tetroxide, uranyl acetate and lead citrate) presents significant cell-structure contrast which has to be discerned from the ND landmarks.

The TEM technique was initially validated on simulated biological slices, comprising NDs dispersed in agarose gel, fixed, resin-mounted, and sliced for TEM. The TEM observations of untransfected cells dispersed with NDs reveal the morphology and distribution, and are consistent with observations made by optical techniques, above. That is, the NDs are loosely agglomerated, and are almost exclusively present in the extra-cellular matrix (Fig. 3a). During this study, some dark features having the morphology of a ND particle were in fact not NDs. That is, it is desirable to confirm that any ND-like feature in fact is a ND, rather than, for example, a nanoprecipitated lead citrate agglomeration. The second approach to confirm that the dark features are indeed NDs is electron diffraction (Fig. 3b) by the ND lattice. Diffraction spots corresponding to diamond are evident over the diffuse scattering of the amorphous mounting medium. The shadow is due to a pointer used to block the primary beam from the camera, to allow acquisition of diffraction patterns with adequate dynamic range. Without this, camera bloom and oversaturation would obscure the fine details.

Discussion: Figure 4 presents a central result of this example. Targeting of the nuclear membrane (Fig. 4a) was accomplished by a covalently conjugated antibody specific to Nup98 (Fig. 4a), which is a nucleoporin (NUP) protein normally found at the nuclear pore, embedded in the nuclear membrane. The particular ND shown in Fig. 4b is composed of three sub-grains (i.e. it is a polycrystalline ND with three domains). High-resolution transmission electron microscopy (HRTEM) (Fig. 4c) reveals lattice fringes of the crystalline diamond structure, and an electron diffraction pattern of the same ND (Fig. 4d) provides further confirmation that the landmark is a ND. This procedure yielded trans fected NDs that were found to be non-agglomerated and attached to the nuclear membrane. The results of Fig. 4 show that TEM can characterize the successful transfection, targeting, and localization (as a landmark or alignment fiducial) of single NDs at a region of interest.

This approach is expected to have a significant transformative impact in the biological sciences, allowing the determination of mechanistic descriptions of biological structures and dynamics. The approach was demonstrated for live-cell labeling, but is expected to be equally powerful for post-slicing labeling, whether by cryo or post-embedding. The intrinsic fluorescence of NDs having NV ^" centers means that no fluorophore is needed for optical imaging of the nanoparticulate markers, meaning that cytotoxic fluorophores and detail- obscuring silver enhancement are unneeded. Transfected and targeted NDs could also serve as image-alignment fiducials for tomographic TEM reconstruction. Last, and importantly, this NV ^" ND approach is amenable to correlated optical and electron microscopy, even in live cell environments over multiple iterations. Methods:

Nanodiamonds, transfection reagents and conjugates. The NDs used in these experiments were prepared by ball milling of larger about 100 μιη diamonds containing NV ^" centers and are on average about 100 nm in diameter. More generally, NDs can have an average diameter or size in the range of about 1 nm to about 900 nm, such as from about 1 nm to about 800 nm, from about 1 nm to about 700 nm, from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about 10 nm to about 250 nm, from about 10 nm to about 200 nm, or from about 10 nm to about 150 nm. In some embodiments nanodiamonds are less than or no greater than 200 μιη in size, less than or no greater than 60 μιη in size, or less than or no greater than 30 μιη in size. The smaller the nanodiamond, the greater its mobility and ability to transport across membranes and tissue. However, NDs that are too small (e.g., less than 10 μιη in size) may be difficult to locate in the TEM images in some embodiments. A good size range to work with is 30-100 μιη in some embodiments.

NDs can have a variety of shapes, such as spherical, spheroidal, and other regular and irregular shapes. Conjugation of NDs to antibodies was performed by oxygen termination of the surface using strong acid treatment followed by EDC (carbodiimide) conjugation to the antibodies. Polypropylene imide dendrimers were purchased from SymoChem (Holland) and were conjugated with maltotriose to address cytotoxicity.

Cells and transfections. All cells used were HeLa cells grown in DMEM with 10% FBS, 1% P/S. Transfections were performed by mixing transfection reagents with ND conjugates in HEPES buffer, or NDs alone in HEPES. Reagents were allowed to precipitate for 20 min at room temperature, followed by drop-wise addition of the solution to cells in serum- free DMEM, 1% P/S for 6 hours. Afterward, the media was replaced with growth media and cells were left to grow for 18-24 hours on plastic coverslips, after which cells were fixed with 2% PFA, and some stained with DAPI. Coverslips were mounted onto slides in 10% PBS/90%> Glycerol and sealed.

Fluorescence microscopy. Confocal microscopy was performed on a Leica SP5-STED microscope. Excitation was done using two laser lines at 514 nm and 548 nm. Samples were bleached by repetitive scanning in order to reduce background. On this system, fluorescence can be detected using a photomultiplier tube (PMT) or avalanche photodiode (APD). Lifetime imaging was performed on a Leica SP2-FLIM microscope with Becker and Hickl SP-830 imaging hardware. Lifetime excitation was performed with infrared light from 900- 910 nm with a tunable TI sapphire laser. In general, however, the NV ^" color center can be excited over a wide range of wavelengths at room temperature (400-1100 nm), with varying amounts of efficiency. Green light excitation can be used.

TEM sample preparation. The cell monolayers grown on coverslips were immersed in a solution of 0.1 M PBS, pH 7.4 containing 2% glutaraldehyde and 2% paraformaldehyde at room temperature for 2 hours, and then at 4°C overnight. The cells were subsequently washed in 0.1 M PBS buffer and post-fixed in a solution of 1% Os0 ₄ in PBS, pH 7.2-7.4. Samples were then buffered in Na acetate, pH 5.5, and stained in 0.5% uranyl acetate in 0.1 M Na acetate buffer, pH 5.5, at 4°C for 12 hours. The samples were sequentially dehydrated in graded ethanol (50%, 75%, 95%, 100%) and infiltrated in mixtures of Epon 812 and ethanol (151 ratio) and 2: 1 for two hours each time. The cells were then incubated in pure Epon 812 overnight and subsequently embedded and cured at 60°C for 48 hr. Sections of 70-90 nm thickness (gray interference color) were cut on an ultramicrotome (RMC MTX) using a diamond knife. The sections were deposited on carbon-film copper grids and double stained in aqueous solutions of 8% uranyl acetate at 60°C for 25 min, and lead citrate at room temperature for 3 min prior to TEM. (Vitreous ice sample preparation also works well, but this technique was not used in this particular example.)

TEM sample examination. TEM examination was performed using an FEI Titan operated at 300 keV. Locations of regions of interest were recorded in relation to a fiducial on the copper grids, allowing location of the same area in subsequent optical microscopy experiments. Low electron beam currents, typically less than 0.6 nA, along with standard low-dose imaging techniques, were employed in order to preserve specimen integrity. For low-magnification imaging, the FEI Titan uses the projector lenses for magnification, the optical path of which blocks many diffracted beams, rendering some NDs visible as dark spots (among real-world artifacts that may also appear dark). At moderate to high magnifications, a restrictive objective aperture is used to block diffracted beams of the nanodiamond, rendering it dark against a relatively lighter background of the embedded cell. Tilting of an eucentric-height positioned sample was occasionally employed to achieve "twinkling" of ND particles as they moved through strongly diffracting (near-zone axis) orientations. HRTEM images were acquired as close to zero defocus as possible in order to minimize the contribution of the amorphous background, which would otherwise obscure visible lattice fringes.

The solution to locating NDs by TEM involves (1) use of a restrictive objective aperture to block substantially all diffracted beams from NDs, making them appear darker than surrounding material, and (2) exploiting their crystalline nature to discern them from sample-preparation artifacts. HRTEM imaging of the lattice fringes of NDs embedded in the amorphous mounting medium is feasible, at near zero defocus of the objective lens.

Confocal Fluorescence Microscopy Control. The bright fluorescence of NDs yields excellent cellular labels. Confocal fluorescence microscopy reveals that control (untransfected) cells incubated with an excess of NDs primarily form aggregates of NDs that remain outside cells, or are occasionally internalized and retained near cell surfaces (Fig. 5b). This interpretation is supported by Y-Z reconstruction slices of the confocal data (Fig. 5c), with bright aggregates, appearing at cell boundaries, or more rarely inside the cells and likely trapped in endosomes. Few, if any, NDs are released into the cytoplasm, and they do not generate sufficient contrast to be discerned from cellular auto-fluorescence. Even in images collected using an avalanche photodiode (APD) (Fig. 5d) capable of detecting single photons, auto-fluorescence of artifacts and cell structures are difficult to discern from NDs. Thresholding of the confocal images and the presence of an artifact in a control sample containing no NDs at the same threshold as that involved to detect NDs (Fig. 5a) show that methods to identify artifacts and NDs beyond spectral filtering and single photon detection can be employed.

TEM Control. The TEM technique was initially validated on simulated biological slices, comprising NDs dispersed into agarose gel, fixed, resin-mounted, and sliced for TEM. Low-magnification imaging (Fig. 6a) reveals a nominally featureless slice, and shows the 125 μιη field-of-view provided by the copper support grid, which is adequate to capture several HeLa cells within one window. Although several dark features are visible in a moderately magnified image of the central region of the same sample (Fig. 6b), many are non-crystalline in nature (preparation artifacts existing in real-world specimens). A single ND (Fig. 6c) imaged in a high diffraction contrast mode shows contrast consistent with a crystalline nanoparticle. Confirmation (Fig. 6d) is obtained in a high-resolution (HRTEM) image, taken near zero defocus, in which lattice fringes of the crystalline ND are evident. Example #2

A researcher working on a therapeutic compound would like to determine the subcellular targeting efficiency of the compound to a particular organelle, in this case the mitochondria. To visualize the compound, it should be conjugated with a marker, and in this case it should be a particulate marker because TEM is involved to achieve the required magnification for visualization. A marker system that makes use of nanodiamonds (NDs) containing fluorescent color centers is chosen. In order to avoid problems of steric hindrance commonly observed with large particulate markers, the NDs are less than 200 μιη in diameter, less than 60 μιη in diameter, or less than 30 μιη in diameter. The NDs in the system chosen in this example contain nitrogen-vacancy (NV ^~ ) color centers because these fluoresce strongly under appropriate laser illumination, thus making it possible to use super- resolution optical techniques to observe uptake and to decide when fixation is appropriate. Diamonds can contain many types of color centers. NV ^" is used, but any other fluorescing diamond color centers can be used. For the researcher's final analysis, TEM is used to locate the NDs within the cytoplasm and in this case to directly observe all NDs in a slice of the cell culture, and to record the position of each one, and its proximity to or association with (in this case) mitochondria. The particular mode of TEM imaging used in this case is that of a restrictive objective aperture that blocks all diffracted beams from the NDs, and a confirmation step of briefly observing each suspected ND in diffraction mode to confirm that it is indeed a ND. Other TEM techniques, both existing and future that may similarly allow distinguishing NDs from cell and mounting matter may be incorporated into this researcher's protocol. The mode of TEM imaging chosen uses the difference in angular distribution of electron scattering by the NDs and the cells or mounting medium to provide, making use of this difference in real or reciprocal space to obtain contrast differences, diffraction patterns, etc. The approach has allowed the researcher to gain statistics that are insightful for the work.

Example #3

A researcher would like to mark a sample post-embedding and post-slicing with NDs. In this case, a transfection agent is not involved, because particles do not penetrate (very deeply, due to steric hindrance) into resin-fixed or frozen samples. They do penetrate somewhat into thawed slices, but that is not the case in this example. One reason for application of the approach in this modality is to achieve a higher concentration of ND markers on the sample. That is, to avoid any potential steric interference of NDs with sub- cellular processes, which could occur if a high concentration of NDs were transfected into a living cell. It is noted that the concentrations used in the above examples were relatively low (e.g., much less than 1% by volume), and are not considered to have affected any subcellular processes. In this example, the researcher applies a ND-containing suspension having a concentration of less than 25% by volume, less than 10% by volume, or less than 3% by volume (although higher concentrations could be used in other examples). The researcher applies the ND suspension, and then proceeds with analyses using optical and/or electron microscopies as described in the detailed description.

Example #4

In a first instance, a researcher is studying temperature changes inside a cell using NV ^" centers in NDs by performing ODMR measurements on the electron spin resonance of the NV ^" center inside live cells. (Temperature measurements can be done with NV ^" centers by monitoring the zero field splitting.) In a second instance another researcher is probing local magnetic fields using NV ^" centers in a cell or tissue. In a third instance, fluorescent NDs are used for tracking purposes (NDs are possibly targeted, but not necessarily so). In all three cases, optical imaging is performed using fluorescent centers. And in all three cases, the researcher may be interested to know the nature of the immediate environment in the vicinity of the NDs, in order to correlate the readout with local cell structure and ultimately, determine function of the cell / tissue. The biological samples can then be imaged by electron microscopy in order to obtain detailed structural images of the local environment near the NDs. The method described herein allows the rapid localization of NDs and the rapid alignment of the electron microscope and its field of view over the region of interest, in order to obtain detailed images while minimizing exposure of the biological samples to the electron beam and minimize damage.

Example #5

A researcher would like to correlate electron and optical images precisely, so that the same region within the same cell is imaged with both modalities. Correlated imaging enables experimentalists to combine the strengths of multiple imaging modalities. The nanodiamonds can be used as in-cell probes for correlation of electron microscopy and optical images. The images in Figures 7 and 10 are of the same diamond cluster inside of cells, as imaged under cryoelectron microscopy and later after dehydration under optical microscopy, though dehydration was not required in this case. The fluorescence intensity profile (Figures 8 and 9) shows a Gaussian profile consistent with diamond fluorophores centered at 1.7μιη with width of ~1μιη in the vertical line region (purple) and centered at 2.4μιη with width of ~1.4μιη in the horizontal line region (green). These values are consistent with the measured values for the diamond cluster in the cryoelectron microscopy image (Fig. 10). Figure 9 shows fluorescence intensity profiles exhibiting a plateau in the peak intensity approximately one micrometer high. Figure 10 shows a cryoelectron microscopy image of the same diamond complex taken prior to optical measurements. Figure 11 shows the optical fluorescence image (zoom-in with respect to Fig. 7) of the same nanodiamond cluster as shown in Figure 10. Thus, Figures 10 and 11 are the sought correlated electron and optical images, respectively.

Example #6

This example describes accessories for correlated optical and electron microscopies and demonstrates excellent quality images of subcellular regions using electron microscopy and localization of nanodiamonds within cells.

Accessories developed for electron and optical imaging

Figures 12 and 13 demonstrate accessories developed for high resolution imaging of mammalian cells and the growth of cells onto these platforms for imaging. The model shown in Figure 12 (generation #1 holder) was fabricated using a 3D printer. Several of these parts were fabricated and used routinely in experiments. The main limitation of the generation #1 system is that transmission-mode optical imaging is difficult because the base is solid (optically opaque). On the other hand, reflective imaging is possible. However, phase contrast transmission mode imaging is helpful in identifying the important features and evaluating the biology. To this end a generation #2 system was designed with the center holding the TEM grid.

Figure 13 shows a photograph of the grid holders. To the bottom left quadrant of the picture, two white plastic holders can be seen. These two pieces were 3D printed according to the CAD drawing of Figure 12. Immediately below the razor blade, a large rectangular object can be seen, which consists of a clear cover slip with parafilm wrapped around the edges. This is the generation #2 system. The parafilm is supporting a TEM grid (copper- colored, disc-shaped object in the center). This assembly enables both TEM and optical imaging (including transmission optical imaging). Prior to this disclosure, it was challenging to image TEM grids on an optical microscope. In order to remove the TEM grid, the parafilm can be completely removed, leaving only a flat coverslip that is wide enough for the grid to slide to the edge where it can be grabbed using tweezers. The user/experimentalist should be careful to avoid running the grid into any bumps, otherwise the grid may bend, leading to dramatically lower image quality.

The razor blade is useful to the generation #2 system, as the parafilm can be completely cleared leaving only a flat coverslip so the grid can be grabbed, while avoiding running the grid into bumps. Aside from holding things together, the parafilm provides a hydrophobic surface to allow a small amount of water to sit over the grid much like a tissue culture chamber with an open air top.

A generation #3 system uses a thinner cover slip (such as by etching a cover slip) and other accessories (such as temperature control, flow ports or microwave antenna for ODMR experiments utilizing NV- color centers in diamond). The generation #2 system enabled separation injections to look at nanodiamonds and then inject the substrate and watch the effect occurring in real time, as driven by syringe, in a STED-compatible format (STED: stimulated emission depletion, a super-resolution optical imaging technique). The cover slip and its composition and dimensions are important to the optical imaging.

Protocol detailing directions to construct a grid holder:

The grid holder allows the removal of the TEM grid from the cell culture. This overcomes an important challenge whereby TEM grids cannot practically be removed from cell culture dishes due to their flat profile.

1. A 2 cm ² square coverslip was first sterilized with a 7:3 ethanol to water mixture.

2. A strip of 10 cm x 2.5 cm of parafilm was cut and sterilized.

3. The glass coverslip was wrapped in the parafilm without stretching the parafilm.

4. To seal the parafilm, it was pressed against a flat surface in order to seal the wax layers together.

5. Parafilm is used to create a hydrophobic surface to hold media in a liquid drop protecting cells.

6. A razor blade was used to cut a square window in both the top and bottom of the parafilm, leaving a 0.5 cm border around the edges of the coverslip. Grid seeding protocol for growing HE 293 cells on quantifoil TEM grid in a six well tissue culture dish. Grid Seeding Protocol for Six Well Dish:

1. 293 Cells were grown to 70% confluency in two wells of a six well dish, seeded from the day before.

2. Before working with the cells a TEM grid holder was first constructed.

Directions to construct a grid holder are as follows. The grid holder allows the removal of the TEM from the cell culture. As TEM grids cannot practically be removed from cell culture dishes due to their flat profile.

• A 2 cm ² square covers lip was first sterilized with a 7:3 ethanol to water mixture.

• A strip of 10 cm x 2.5 cm of parafilm was cut and sterilized.

• The glass coverslip was wrapped in the parafilm without stretching the parafilm.

• To seal the parafilm it was pressed against a flat surface in order to seal the wax layers together.

• A razor blade was used to cut a square window in both the top and bottom of the parafilm, leaving a 0.5 cm border around the edges of the coverslip.

3. On one of the wells of the six well plate. The media was replaced with 2 mL of DMEM media (containing 10% FBS, 1% PS). The grid holder was then added to this well, taking to leave no air bubbles on the underside of the grid holder.

4. To the well with the grid holder the TEM grid was added to the grid holder.

5. On the other well the media was removed and 0.5 mL of PBS was added.

6. The PBS was removed after 1 minute and 0.5 of accutase was added to the well to remove the cells from the bottom of the dish.

7. The cells were then incubated at 37 ⁰ C for two minutes.

8. 1 mL of DMEM was then added to the six well and the entire media was pipetted against the bottom of the dish to insure the cells are unclumped.

9. 400 of suspended cells was added to the well with the TEM grid.

10. The cells were then allowed 24 hours to attach to the TEM grid.

The results are shown in Figures 15-27. Example #7

Illuminating nanodiamonds: Luciferase was used to illuminate the nanodiamonds, which results in excellent excitation transfer efficiency, as shown in Figures 28 and 29. One advantage of such illuminating nanodiamonds is that the use of a laser to generate fluorescence is no longer necessary, thereby reducing damage to the tissues and cells in the test sample. Instead, bioluminescent NV- nanodiamond probes are used.

While certain conditions and criteria are specified herein, it should be understood that these conditions and criteria apply to some embodiments of the disclosure, and that these conditions and criteria can be relaxed or otherwise modified for other embodiments of the disclosure.

While the invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention as defined by the appended claim(s). In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, operation or operations, to the objective, spirit and scope of the invention. All such modifications are intended to be within the scope of the claim(s) appended hereto. In particular, while certain methods may have been described with reference to particular operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or re-ordered to form an equivalent method without departing from the teachings of the invention. Accordingly, unless specifically indicated herein, the order and grouping of the operations is not a limitation of the invention.