GB2480640A - Luminescence enhancing beads for bio-imaging - Google Patents

Luminescence enhancing beads for bio-imaging Download PDF

Info

Publication number
GB2480640A
GB2480640A GB201008768A GB201008768A GB2480640A GB 2480640 A GB2480640 A GB 2480640A GB 201008768 A GB201008768 A GB 201008768A GB 201008768 A GB201008768 A GB 201008768A GB 2480640 A GB2480640 A GB 2480640A
Authority
GB
United Kingdom
Prior art keywords
poly
light
use
deflective
beads
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
GB201008768A
Other versions
GB201008768D0 (en
Inventor
Nathalie De Vocht
Anne-Marie Van Der Linden
Peter Ponsaerts
Zwi Nisan Berneman
Geofrey De Visscher
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universiteit Antwerpen
Original Assignee
Universiteit Antwerpen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universiteit Antwerpen filed Critical Universiteit Antwerpen
Priority to GB201008768A priority Critical patent/GB2480640A/en
Publication of GB201008768D0 publication Critical patent/GB201008768D0/en
Publication of GB2480640A publication Critical patent/GB2480640A/en
Application status is Withdrawn legal-status Critical

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/645Specially adapted constructive features of fluorimeters
    • G01N21/6456Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458Fluorescence microscopy
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6486Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10Investigating individual particles
    • G01N15/14Electro-optical investigation, e.g. flow cytometers
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N2015/0038Investigating nanoparticles

Abstract

An optical enhancer system comprises fluid filled beads that alter the optical properties of cells that engulf the beads resulting in enhancement of a non bead related bioluminescent signal. The beads have low light scattering properties and when added to a high scattering medium they enhance light travel in a direction toward a light detector rather than scatter light away from a detector. The beads or particles comprise microspheres with an outer membrane and an inner core of low light scattering medium such as an optically translucent solution or gas. The optical enhancer beads are used for bioluminescence measurements of cells both in vivo and in vitro. Larger samples or fewer cells may be imaged as the luminescent signal is more focused on the detector.

Description

Bio Imaging Beads

FIELD OF THE INVENTION

The present invention relates generally to biolumiscence measurements of cells and more particularly to localization and other functional measurements both in vivo and in vitro of these cells. It is based on the finding that the presence of particles, in particular microspheres, with low light scattering properties, i.e. lower light scattering properties compared to the light scattering properties of the surrounding medium, -hereinafter also referred to as light-deflecting particles -in a high scattering medium (HSM) results in the amplification, enhancement of a luminescent signal within said HSM. It accordingly provides the use of such light-deflecting particles, in particular microspheres, in the amplification / enhancement of a luminescent signal within a high scattering medium, in particular in the amplification / enhancement of a bioluminescent signal.

BACKGROUND OF THE INVENTION

In the current and developing world of cell transplant there is an obvious need to obtain infonTnation such as location, migration, functionality, viability, phenotype, etc. from the cell graft in a non-destructive way.

The actual state of the art already provides a set of techniques each enabling to acquire some information from the cell grafts in a either a destructive or non destructive manner, yet there is also the need to broaden this technical base to enable working with smaller samples and/or enhance low signals.

A fairly simple yet destructive method is direct labelling of cells with vital dyes prior to implantation. For example dyes such as but not limited to 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (Dii) or its relatives DiO, DiD, DiA, and DIR incorporate in the cell membrane and can be visualised by means of fluorescent microscopy'. If one knows the fate of implanted cells they can also be visualised with

I

---

immunohistochemisty and microscopy. Unfortunately these techniques require sectioning of the tissue and are therefore destructive to the tissue and recipient organism.

An extremely powerful, yet expensive method to follow cells upon in vivo implantation is labelling with iron containing beads which are visualised by magnetic resonance imaging. These superparamagnetic beads, which act as T2 contrast agents2'3, can be divided in 3 classes according to their size3. USPIO's or ultrasmall superparamagnetic iron oxide particles are approximately 10-20 nm in size.

Superparamagnetic iron oxide particles or SPIO's range from 50 to 100 nm and the micron-size iron oxide particles or MPIO's range from 1 up to 6 pm. The beads all provide a high MR sensivity with USPIO's and SPOT's enabling detection of a few cells and MPIO's even providing single cell resolution4.

These iron oxide containing beads are widely used for cell labelling because they have the following properties: a high signal per unit of metal, biocompatibility, biodegradability, the dextran coating allowing functionalisation of the surface and detection of the beads with post mortem light or electron microscopy5. These beads require cellular uptake which is a prerequisite not suitable for all celltypes because the efficiency of internalisation is phenotype dependent. Beads sticking to the surface might hinder cellular interactions and are potentially transferable to other cells2'6. The labelling strategies for these beads are either by intravenous injection, in vitro labelling or in situ labelling. Intravenous injection, the first labelling method reported, causes the beads to be taken up by phagocytosis in the body35. To acquire a more targeted cell labelling this was later done in vitro by exposing selected cell (such as but not limited to mesenchymal stem cells) to a particle suspension prior to implantation3. Finally, in situ labelling can be used to track mobile cells out of an

immobile background population7.

The labelling can also be facilitated by by techniques such as but not limited to electroporation, magnetoporation, dextran coat modification, alternative coating, transfection agents, antibodies In this age of gene transfer it is also obvious that iron metabolism modulating genes have also been used for labelling cells with the purpose of MRI detection. A first example is the transfer with viral vectors of the ferritin gene into the target cells.

Ferritin8 is a highly conserved protein that is ubiquitous both within and over the animal species. Tt is responsible for controlled storage and release of iron, which is both a necessary element but also potentially cytotoxic. The protein assembles hollow spheres out of 24 monomers and stores up to 4500 iron atoms as oxy-hydroxide in its center. The ferritin particle is approximately 1 mm in diameter and the iron core 5.5nm. This makes them comparable to USPIO's in size. The MagA gene is another example currently under study for these purposes. This gene is part if the genetic program necessary for magnetotactic bacteria to construct magnetosome, a subcellular structure used for orientation. Gene transfer of this gene has recently also been used as a MRI contrast agent9. The gene list mentioned here are not exhaustive, but merely

examples.

Although these magnetic beads and magnetic reporter genes can be used to label various cells and tissues such as but not limited to neural stem cells4 or endogenous neural stem cells in brain tissue'° and mesenchymal stem cells in heart tissue", it also has a major drawback. The equipment or more precisely the MR scanners required to image the labelled cells and surrounding tissue are extremely expensive and often require lengthy procedures for imaging. In addition the iron oxide create inhomogeneities leading to a signal many times their own size, hereby the image is largely overestimating the presence of cells in a specific area'2.

Another labelling strategy is based on the transgenic expression of fluorescent proteins. Although different hues, from either other species or by mutations, are available the most commonly used is green fluorescent protein derived from a gene from the jellyfish Aequorea Victoria (Murbach and Shearer, 1902). The reporter gene has been successfully transfere to both pro and eukaryotes including human cells and have for example been used to study cell survival of transplanted bone marrow derived stromal cells'3. Although being less phototoxic than for example FITC, the fluorescent proteins are hampered by the fact that both the exciting and emitting photons need to travel through the tissue for detection. Either the species need to be relatively translucent (e.g. zebrafish) or sectioning is required to obtain good image quality.

One step further is to transfer a reporter gene of which the protein produces light without being first excited by another photon. The most commonly used reporter gen codes for firefly (Photinus pyralis) luciferase, although other recombinant luciferases (e.g. Renilla sp.) are available. Trangenic cells expressing the luciferase gene will upon administration of luciferin produce light. This technique is particularly interesting because it allows imaging of luciferase expressing cell non-invasively in a living whole organism by means of a CCD camera. This technique has been shown usefull to follow up for example transplanted pancreatic islets'4, MAPC's'5, mesenchymal stem cells'6, embryonic stem cells'7 and many more. More specifically it has been usen in cancer research to study tumor growth'8 and in neuroscience to visualise transplanted bone marrow-derived stromal cells in brain tissue'9. Here again there are some provisos and prerequisites towards the use of this technique. Due to scattering properties of the tissue as well as the absorption of the produced photons the size of the animal is limited and the amount of cells needed relatively large.

Furthermore the scattering and absorption properties are dependent on the tissue and are generally not linear.

The final example of state of the art technology relates to bioluminescence resonance energy transfer or BRET. It is strongly related to some of the aforementioned examples because it is based on the efficient resonance energy transfer between a bioluminescent donor moiety and a fluorescent acceptor moiety. For BRET the photons produced by luciferase are transferring their energy to a near by, typically 10 to 100 angstrom, receptor. The photons are here accepted and light with a lower energy is emitted. This optical technique was originally designed to be used to study molecular interactions, however cell tracking has also been performed20.

Because of the importance of cellular information in the upcoming age of cell therapy, even nanoparticles combining the aforementioned principles have been developed recently. Quadruple imaging modality nanoparticles have been reported combining fluorescence, bioluminescence, BRET, PET and MR The above list of cell tracking techniques is covering widely used techniques in cell biology, imaging and tissue engineering/regeneration, but the list is not exhaustive and therefor incomplete.

As is obvious from the enunciation above need for technical improvement is apparent.

In the field of optical imaging solutions the absorbtion and scattering of the tissue22 are causing loss of signal and focus quit rapidly. Consequently the recipient needs to be small or translucent and/or the transplanted cells or tissue rather large. Also the scattering causes blurring of the signal thereby exaggerating the actual location of the graft.

The present invention adds to the previous bioluminescence applications by providing an optical enhancer system comprising microspheres that alter the optical properties of the cells resulting in the amplification of a non-bead related bioluminescent signal.

As will be apparent from the examples hereinafter, this potentiates the optical technique, i.e. the application of the non-bead related bioluminescent signal by allowing the use of larger animals, less cells and focussing the signal.

SUMMARY OF THE INVENTION

The invention provides optical enhancer beads for use in biological, medical and biotechnological applications, and is based on the finding that the presence of particles, in particular microspheres, with low light scattering properties, i.e. lower light scattering properties compared to the light scattering properties of the surrounding medium, -hereinafter also referred to as light-deflective particles -in a high scattering medium (HSM) results in the amplification, enhancement of a luminescent signal within said HSM.

It is accordingly a first aspect of the present invention to provide the use of light-deflective particles, in the amplification or enhancement of a luminescent signal within a high scattering medium. As the optical enhancement was primarily observed in biological media, including both in vitro assays and in vivo models, in a particular embodiment the present invention provides the of light-deflective particles, in the amplification or enhancement of a bioluminescent signal.

Within the aforementioned biological media said bioluminescent signal is typically created by exogenous and endogenous sources; in particular exogenous sources selected from transgenic expression of fluorescent or bioluminescent reporter genes; dyes; antibodies; nanobodies; or nanoparticles. It is accordingly a further objective of the present invention to provide the use of the light-deflective particles in the amplification or enhancement of a bioluminescent signal created by exogenous and endogenous sources; in particular exogenous sources selected from transgenic expression of fluorescent or bioluminescent reporter genes; dyes; antibodies; nanobodies; or nanoparticles.

As used herein the light-deflective particles have no specific shape requirements as long as the particles comprises a surface area sufficient to create a light "focussing" effect, due to the deflection of light by the particle, which effect is visually perceptible to a viewer observing an enhancement of the luminescent signal within a HSM.

Satisfactory effects have been obtained by using particles having light deflecting surface areas of from between and about 3x10'° mm2 to 0.25 mm2; in particular from between and about 3x10'° mm2 to 0.1 mm2; more in particular from between and about 3x101° mm2 to 1x103 mm2.

In a particular embodiment, the light-deflective particles have a length of from about nm to 400 microns (0.00003 -0.4 mm), a width of from about 10 nm to 400 microns (0. 0000 1-0.4 mm) and a thickness of 10 nm to 20 microns (0.0000 1 -0.02 mm). In an even further embodiment the light-deflective particles are microspheres with a diameter of between and about 10 nm to 200 microns (0.00001 -0.2 mm), in particular with a diameter of between and about 10 nm to 20 microns (0.0000 1 -0.02 mm), more in particular with a diameter of between and about 10 nm to 200 nm (0.0000 1 -0. 0002 mm), within said embodiment the light-deflective microspheres are hereinafter also referred to as optical enhancer beads.

In a further embodiment of the aforementioned application, the light-deflective particles are further characterized in that they comprise an outer membrane and an inner core. Particularly, and for use in the biological media, the outer membrane is made from an inert bio-compatible material, such as for example glass, ceramics, bio-compatible metals, bio-compatible polymers, or biodegradable materials.

As used herein bio-compatible materials generally refer to materials that do not promote tissue adhesion and do not inhibit cellular growth and which are not otherwise toxic to living systems. Bio-compatible polymers are for example selected from the group consisting of polystyrene, polyacrylic acid, silicone, polypropylene, polyester, polytetrafluoroethylene, polyethylene terephthalate, polyurethane, polymethylmethacrylate, and polymers of ethylenediamine, diethylenetriamine, allylamine or hydroxyethylmethacrylate.

Alternatively, the outher membrane of the light-deflective particles may be coated with substances that improve the biocompatibility of the particles, such as but not limited to dextran, citrate, dimercaptosuccinic acid.

In a particular embodiment the light-deflective particles used within the context of the present invention are further characterized in that the outher membrane is functionalised by protein or peptide coating. Said proteins or peptides can be any one or more of, -antibodies or nanobodies; -ligands to membrane bound moieties such as but not limited to proteins, glycoproteins, phospholipids or any fraction thereof -fluorescent proteins; -light emmiting proteins such as but not limited to firefly or renilla luciferase; or -the fluorescent or light emmiting domain of the relevant light emmiting proteins.

In a second aspect the present invention provides light-deflective particles as defined hereinbefore, for use in the amplification or enhancement of a luminescent signal within a high scattering medium, said particles comprising an outer membrane and an inner core, and being characterized in that the inner core at least partially consists of a low light scattering medium such as an optically translucent solution, solid or gas.

In principle any optically translucent solution can be used in the aforementioned particles and is for example selected from the group but not limited to water, saline buffers, optical gels, glass. In a particular embodiment the optically translucent solution is combined with any one or more of -fluorescent compounds such as but not limited to fluorescein isothiocyanate, rhodamine, coumarin, sulforhodamine 101 acid chloride, ethidium bromide, propidium iodide or 4',6-diamidino-2-phenylindole; -ion indicators such as but not limited to the Ca++ dependent dyes FURA-2, FURA- 2AM or INDO-1; -magnetic indicators for MRI.

As already mentioned hereinbefore, in one aspect of the present invention, the outer membrane of the reflective particles is made from a bio-compatible material; in particular biodegradable materials.

As used herein biodegradable refers to any materials that are capable of being completely degraded and/or eroded when exposed to bodily fluids such as blood and can be gradually resorbed, absorbed and/or eliminated by the body. The processes of breaking down and eventual absorption and elimination of the material can be caused by, for example, hydrolysis, metabolic processes, bulk or surface erosion, and the like.

In some embodiments, very negligible traces or residue may be left behind. Whenever the terms "degradable," "biodegradable," or "biologically degradable" are used in this application, they are intended to broadly include biologically erodable, bioabsorbable, and bioresorbable materials, such as for example liposomes, biodegrable polymers and biodegradable polysaccharides, as well as other types of materials that are broken down and/or eliminated by the body.

Liposomes as used herein are for example composed of naturally derived phospholipids with mixed lipid chains (like phosphatidylethanolamine and phosphatidyicholine), or of pure surfactant components like DOPE (dioleoylphosphatidylethanolamine). In a particular embodiment liposomes are constructed with PEG (Polyethylene Glycol) studding the outside of the membrane.

The PEG coating, which is inert in the body, allows for longer circulatory life for the drug deliveiy mechanism. These liposomes are known as "stealth liposomes", and research have been able to allow liposomes to avoid detection by the body's immune system, specifically, the cells of reticuloendothelial system (RES). In addition to a PEG coating, most stealth liposomes also have some sort of biological species attached as a ligand to the liposome in order to enable binding via a specific expression on the targeted drug delivery site. These targeting ligands could be monoclonal antibodies (making an immunoliposome), vitamins, or specific antigens.

Liposomes, usually but not by definition, contain a core of aqueous solution; lipid spheres that contain no aqueous material are called micelles, and also within the admit of the present invention.

Inert biodegradable polymers and polysaccharides are for example selected from the group consisting of poly(alkylene glycol), poly(2-hydroxyethyl methacrylate), poly(3-hydroxypropyl methacrylamide), hydroxylated poly(vinyl pyrrolidone), sulfonated dextran, sulfonated polystyrene, fibrin, fibrinogen, cellulose, starch, collagen, hyaluronic acid, heparin, a graft copolymer of poly(L-lysine)-graft-co-poly(ethylene glycol), poly(L-lactide), poly(D,L-lactide), poly(glycolide), poly(L-lactide-co- glycolide), poly(D,L-lactide-co-glycolide), poly(caprolactone), poly(L-lactide-co- caprolactone), poly(D,L-lactide-co-caprolactone), polyhydroxyalkanoates, poly(3- hydroxybutyrate), poly(4-hydroxybutyrate), poly(hydroxyvalerate), poly(3 -hydroxybutyrate-co-valerate), poly(4-hydroxybutyrate-co-valerate), poly(ester amides), poly(anhydrides), poly(carbonates), poly(trimethylene carbonate-co- glycolide), poly(trimethylene carbonate-co-L-lactide), poly(trimethylene carbonate-co-D,L-lactide), poly(dioxanone), poly(phosphazenes), poly(orthoesters), poly(tyrosine-co-carbonates), polyalkylene oxalates, poly(glycerol-co-sebacic acid esters), cyanoacrylates, poly(amino acids), poly(lysine), poly(glutamic acid) and mixtures thereof In a third objective the present invention provides a method of amplifying or enhancing a luminescent signal within a high scattering medium, said method comprising adding a luminescent enhacing amount of light-deflective particles as defined hereinbefore, to said high scattering medium. In a particular embodiment said method is applied for amplifying or enhancing a bioluminescent signal; more in particular bioluminescent signal created by exogenous and endogenous sources; even more in particular created by exogenous sources selected from transgenic expression of fluorescent or bioluminescent reporter genes; dyes; antibodies; nanobodies; or nanoparticles.

Within the context of a biological environment said method is either used; -for diagnostic in vivo imaging; and the method comprises administering to a subject a luminescent enhacing amount of deflective particles as defined herein; -for diagnostic in vitro imaging; and the method comprises administering to an in vitro assay a luminescent enhacing amount of reflective particles as defined herein; -for monitoring bioreactors; and the method comprises administering to a bioreactor a luminescent enhacing amount of reflective particles as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein: Figure 1 This schematic diagram illustrates several beads that have the enhancing effect of which the most generic is the bead comprised out of an outer polystyrene shell encasing an interior medium having low scattering properties. The interior medium can be a gas, fluid or solid. As shown in the three additional descriptions the generic beads can be altered with several additional marker thus combining other properties with the optical enhancer effect. Additional markers such as but not limited to are iron, gadolinium for MRI imaging, radioactive markers for PET & SPECT imaging, Fluorescent markers for microscopy and bioluminescent markers.

Figure 2 The working principle of the optical enhancer beads is shown in this simplified diagram. Light hitting the particles is only partly reflected at its surface. Light entering the particles is only refracted upon entrance and exit. The diagram further shows the behavior of light traveling to a solution and includes for theoretical cases low scattering medium (LSM) or high scattering medium (HSM) each with or without beads. A: shows light traveling through LSM. Since the photons are not deflected the signal freely moves through the medium and reached the detector unaltered. B: shows the same case yet here the beads are added to the medium. Because the beads, having an outer polystyrene shell, are made from a different material as the medium, they will cause scattering of the light. Consequently this results in loss of signal. When replacing the LSM with HSM the properties wil alter. In high scattering properties light will be lost anyway (C), however adding beads to such an environment can recover some of the light, mainly by altering the overall scattering properties enabling more light to reach the detector (D). The enhancer effect therefore is also dependent on the environment in which it is used.

The models are somewhat simplified because (1) it does not account for potential absorbers in the media (2) it is constructed for a homogeneous high or low scattering environment. In reality a cell suspension can be considered a 2 compartment model with a low scatteting medium (LSM) and a high scattering medium (HSM) component, a tissue would rather be a HSM only.

Figure 3 In vitro light intensities measured in control cells (no beads) and cells labeled with glacial blue labeled MPTO's, glacial blue labeled beads and polystyrene beads.

Figure 4 The enhancer effect calculated from the data presented in the previous (Figure 3) figure. The signals of the beads containing cells were controlled for the intensity of the cells without beads. The resulting ratio shows an enhancer effect of approximately 1.5 for glacial blue labeled MPIO's and 1.7 for glacial blue labeled beads and polystyrene beads.

Figure 5 The individual plots of the forward and side scatter of luciferase and GFP transgenic cells. Adding beads clearly alters the scattering properties of the cells as is shown by the increases in side scatter.

Figure 6 Histogram showing the in vivo bioluminescence signal intensity for mice implanted with cells expressing BMSC-Luc/eGFP at week and week 2 post implant (PT) optionally in the presence of GB MPIO beads (black bars). Bioluminescence signal intensity is expressed as photons per second per steredian (phls/sr) or photons/s/sr'cm2.

Figure 7 Enhancer effect of different beads in luciferase containing bone marrow stem cells.

Bioluminescence signal intensity is expressed as photons per second per steredian (phls/sr) or photons/s/sr/cm2.

DETAILED DESCRIPTION OF THE INVENTION

Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating particular embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Instead, the scope of the invention is defined by the appended claims and equivalents thereof As already mentioned hereinbefore, in a particular embodiment of the present invention the optical enhancement was observed in biological media, including both in vitro environments and in vivo models, using light-deflective particles, such as the beads shown in Figure 1.

Compared to the high light scattering surrounding media, the interior of the beads represents a homogenous environment with low light scattering behaviour. As such, light entering the beads will pass freely through the particles and is only refracted upon entrance and exit of the particles (see schematic diagram as shown in figure 2).

Within such high light scattering biological media the beads effectively increase the optical signal observed with a detector such as but not limited to in vivo bioluminescence camera, an in vitro bioluminescence detector, plate readers. The beads are therefore acting as enhancers of signals created by exogenous and endogenous sources. Examples of exogenous sources are transgenic expression of fluorescent or bioluminescent reporter genes, dyes, antibodies, nanobodies, nanoparticles. For endogenous sources the light source is incorporated in the bead architecture but the light is resulting from incorporated sources such as but not limited to fluorescent or bioluminescent molecules, dyes, antibodies, nanobodies, nanoparticles. The beads also can be used to alter light transmission through tissue from an external source as is used by such techniques as but not limited to in vivo spectrophotometry (e.g. near infrared) and in vitro spectrophotometry (e.g. optical monitoring systems for bioreactors, high throughput screening cell assay).

A particular embodiment of present invention is where the optical enhancer beads are taken up by cells to be grafted into a recipient to facilitate transduction of optical signals, for example to be used in phototherapy and optogenetics.

Another particular embodiment of present invention is where the optical enhancer beads are taken up by cells to increase the signal of an in vitro assay Another particular embodiment of present invention is where the optical enhancer beads are not taken op but reside in the medium of the cells and increase the signal of an in vitro assay or monitoring system.

Another particular embodiment of present invention is where the optical enhancer beads' uptake is facilitated by a secondary agent such as but not limited to poly-l-lysine, electroporation, magnetoporation. Because of the above mentioned restrictions and limitation of photon mediated measuring and monitoring techniques we focused on optical properties of beads enhancing the signal progression through a cell containing solution.

The outer shell' or outer membrane' as used herein is preferably made from a biocompatible material, such as for example glass, ceramics, bio-compatible metals, bio-compatible polymers, or biodegradable materials. Where there are no particular requirements regarding the thickness of said outer membrane, its physical properties should be such that it is capable to deflect optical wavelengths (typically in a range from about 380 or 400 nanometres to about 760 or 780 nm) at its surface.

Further to the examples of bio-compatible polymers and of biodegradable materials mentioned hereinbefore, the bio-compatible metals generally refers to any metal that does not promote tissue adhesion and does not inhibit cellular growth and which are not otherwise toxic to living systems. Biocompatible metals are for example selected from but not limited to the group consisting of tantalum, stainless steel, platinum-tungsten alloys, and other similar metals and alloys.

It is also obvious that anyone skilled in the art can envision further functionalisation of the optical enhancer beads either by adding components to the outer shell or to the the inner core material. Further to the funtionalizations mentioned hereinbefore, said functionalisation may consist of adding componenst to the outer shell that enhance the optical reflectivity of the particles. The latter is for example achieved by coating the particles with an inert, tight-deflective pigment or other material. Bio-compatibte, inert, crystalline pigments and materials are preferred. For example, light-deflective particles of the following materials may be utilized: * Copolyester/acrylates copolymers, such as Crystalina 321, 322, 323 and 324 iridescent glitter particles (polyester/acrylic optical core with polyester outer layer), by Meadowbrook Inventions, Inc. of Bemardsville, NJ; * Polyethylene terephthalate; * Mica with titanium dioxide and ferric ferrocyanide; * Mica with titanium dioxide and chromium oxide; * Mica with titanium dioxide and silica; * Polyester fitm; * \Tacuum-metalized polyester film; * Calcium sodium borosilicate coated with titanium dioxide, such as Reflecks platelets by Englehard Corp. of Isselin, NJ; * Mica with iron oxide and titanium dioxide, such as Timiron 7 MP-24 Karat Gold sparkling powder, by EM Industries, Inc. of Hawthorn, NY.

This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter.

Particular embodiments and examples are not in any way intended to limit the scope of the invention as claimed. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

EXAMPLES

Example 1. beads in cells bioluminescence Bone marrow derived stromal cells (BMSC) and BMSC genetically engineered with (i) the Luciferase (Luc) reporter protein or (ii) the Luciferase and enhanced Green Fluorescent Protein (eGFP) reporter proteins were cultured according to our standard protocol'9. BMSC, BMSC-Luc and BMSC-Luc/eGFP were cultured in complete expansion medium' (CEM), consisting of Iscove's modified Dulbecco's medium (IMDM, Cambrex, East Rutherford, NJ, USA) supplemented with 8% foetal bovine serum (FBS; HyClone Laboratories, Logan, Utah, USA), 8% horse serum (HS; nvitrogen, Merelbeke, Belgium), 1 OOU/ml penicillin + 100 mg/ml streptomycin (Invitrogen, Merelbeke, Belgium) and O.25mg/ml amphotericin B (Invitrogen, Merelbeke, Belgium). For BMSC-Luc/eGFP cultures, CEM was additionally supplemented with I tg/ml Puromycine (Invivogen, Toulouse, France). BMSC cultures were harvested twice a week using trypsin-EDTA (Invitrogen, Merelbeke, Belgium) and passaged at a 1:3 ratio in 15 ml CEM in T75 culture flasks.

Fluorescent (Glacial blue) micron-sized particles of iron oxide (GB-MPIO), fluorescent only (GB-FLUO) or polystyrene particles (cat n° MEO4F/7833, FCO4F/8339, PCO4N/9504, Bangs Laboratories, Fishers, IN, USA) were used for in vitro labeling of BMSC-Luc/eGFP. For cell labeling, 5x 106 particles/ml culture medium were added to subconfluent (50-70% confluency) BMSC-Luc/eGFP cultures.

Following overnight incubation (16h), particle labeled BMSC-Luc/eGFP were washed three times with PBS and cells were further cultured for 24h to enable homogeneous distribution of particles.

To confirm uptake of GB MPIO a subsample was evaluated by standard fluorescence microscopy. Therefore, GB MPIO labeled BMSC-Luc/eGFP grown on cover slips were washed twice with PBS, followed by fixation with 4% paraformaldehyde in PBS for 30 minutes at room temperature. Next, cover slips were washed in PBS and demineralized water, followed by mounting (Prolong Gold antifade reagent; nvitrogen, Merelbeke, Belgium). Visualization of GB MPIO labeled BMSC-Luc/eGFP was done using an Olympus BXS 1 fluorescence microscope (Olympus, Center Valley, PA, USA) equipped with an Olympus DP7 1 digital camera. Olympus Ce11F Software (Olympus, Center Valley, PA, USA) was used for image acquisition and processing.

Serial dilutions (5x104 -4x105 cells/well) of BMSC, BMSC-Luc/eGFP and GB MPIO labeled BMSC-Luc/eGFP were prepared in black 96-well plates (Elscolab, Kruibeke, Belgium). Immediately after D-luciferin administration (7.5 tg D-luciferinlml dissolved in PBS; Promega Benelux, Leiden, The Netherlands), the plates were imaged for 5 minutes using a real-time Photon-imager system (Biospace Lab, Paris, France). At the end of every acquisition, a photographic image was obtained. The data were analyzed with M3Vision software (Biospace Lab, Paris, France). Light emission (Figure 3) was measured from a fixed region of interest on each well and values of signal intensity are presented in photons per second per steredian per square centimeter (phls/sr/cm2).

The data are then normalized against the average measurement of cells without beads to estimate the enhancer effect. The three types of beads examined all a slightly different yet always present enhancement effect. The iron containing glacial blue labeled GB-MPIO's showed the least enhancement but enhanced the signal 1.46 ±0.27 times on average. The beads that did not contain the iron, which can act as a light absorber inside the bead, both had a higher enhancement effect. The particle labeled with glacial blue and the polystyrene paricles enhanced the signal 1.66 ± 0.38 and 1.73 ± 0.36 times respectively. See also figure 4 for more detailed information on the enhancer effect. Compared to the control cells (no beads and their intensity taken as 1.0) the resulting ratios show an enhancer effect of approximately 1.5 for glacial blue labeled MPIO's and 1.7 for glacial blue labeled beads and polystyrene beads.

Although some variation does occur in the particles enhancer effect they all possessed this effect and not surprisingly the polystyrene beads which do not contain potential absorbers such as iron and glacial blue were performing the best.

Example 2 Beads in cells scattering To further evaluate the optical effect of the beads when taken up by the cells we performed a fluorescent sorting experiment focusing on scattering properties.

This was performed with BMSC-Luc/eGFP as controls and the same cells after having taken up GB MPTO, GB fluo or polystyrene beads. The harvested cultures were washed once with PBS, resuspended in PBS and directly analyzed using an Epics XL-MCL analytical flow cytometer (Beckman Coulter, Fullerton, CA, USA).

For all measurements, cell viability was assessed through addition of GelRed (lx final concentration; Biotum, Hayward, CA, USA) to the cell suspension immediately before flow cytometric analysis. At least 10x103 cells were analyzed per sample and flow cytometry data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).

Compared to the control cells (no beads -upper left corner of figure 5), cells comprising beads (GB-MPIO -upper right corner in figure 5; GB-fluo -lower left corner in figure 5; polystyrene -lower right in figure 5) have altered scattering properties with an apparent increase in side scattering. Tt accordingly demonstrates that the presence of optical enhancer beads within cells alters there optical characteristics and as is shown in the present examples, results in an enhancement (amplification) of an optical signal present within said cellular environment.

Example 3 In vivo enhancer effect in transplanted neural stem cells.

Protocol Cell preparation for implantation experiments Following harvesting of BMSC-Luc/eGFP and GB MPTO labeled BMSC-Luc/eGFP, cells were washed twice with PBS and resuspended at a concentration of 133x106 cells/ml in PBS. Cell preparations (mean viability of> 95%) were kept on ice until cell implantation.

Cell implantation experiments All surgical interventions were performed under sterile conditions. The mice were anaesthetized by an intraperitoneal injection of a ketamine (80 mg/kg) + xylazine (16 mg/kg) mixture and placed in a mouse stereotactic frame. Cell implantation was reproducibly targeted in the right hemisphere at following coordinates to bregma: 2 mm posterior, 2 mm lateral and 2 mm ventral. For this, a midline scalp incision was made to expose the skull, and a hole was drilled in the skull using a dental drill burr at the given coordinates. Thereafter an automatic micro-injector pump (KD Scientific, Holliston, MA, USA) with a 10 pi Hamilton Syringe was positioned above the exposed dura. A 30-gauge needle (Hamilton, Reno, NV, USA) attached to the syringe, was stereotactically placed through the intact dura to a depth of 2 mm. After 1 minute of pressure equilibration, 3 pi of cell suspension was injected at a speed of 0.7 p1/mm.

Before needle retraction, a waiting period of 5 minutes was kept in order to allow for pressure equilibration and to prevent backflow of the injected cell suspension. Next, the skin was sutured, a 0.9% NaC1 solution was given subcutaneously in order to prevent dehydration, and mice were placed under a heating lamp to recover. During the entire follow-up period, mice were kept in normal day-night cycle with free access to food and water.

In vivo Bioluminescence (BLI) At different time points between day 1 and week 2 post-implantation, mice were analyzed by real-time in vivo BLI in order to determine the presence or absence of viable BMSC-Luc/eGFP and GB MPIO labeled BMSC-Luc/eGFP cell implants in the CNS. For this, mice were anaesthetized by a mixture of isoflurane (Isoflo®) and oxygen (3% induction, 1.5% maintenance), followed by an intravenous injection of D-luciferin (150 mg/kg body weight dissolved in PBS; Promega Benelux, Leiden, The Netherlands). Immediately after D-luciferin administration, mice were imaged for minutes using a real-time Photon-imager system (Biospace Lab, Paris, France). At the end of every acquisition a photographic image was obtained. The data were analyzed with M3Vision software (Biospace Lab, Paris, France), which superimposes the bioluminescence signal on the photographic image. Light emission was measured from a fixed region of interest on the mouse head and values of signal intensity are presented in photons per second per steredian (phls/sr) or photons/s/sr/cm2.

Results In vivo BLI of GB MPIO labeled BMSC-Luc/eGFP In order to investigate the in vivo survival of GB MPIO labeled BMSC-Luc/eGFP, 4x105 unlabeled (n = 15) or labeled (n = 15) BMSC-Luc/eGFP were implanted in the CNS of immune competent mice. In vivo survival of both cell populations was non-invasively monitored by BLI (data not shown). For unlabeled BMSC-Luc/eGFP, a clear BLI signal was observed in 11/12 mice analyzed at week 1 post-implantation and in 6/11 mice analyzed at week 2 post-implantation. For GB MPIO-labeled BMSC-Luc/eGFP, a clear BIJ signal was observed in 14/14 mice analyzed at week 1 post-implantation and in 10/13 mice analyzed at week 2 post-implantation. The detection of a clear BLT signal (i.e. signal intensity within region of interest at least 25 times higher as compared to a control region of interest) was considered to indicate the presence of a viable cell graft.

Interestingly, further statistical analysis of BLI signals detected in mice with viable cell grafts revealed a higher BLI signal intensity for grafted GB MPIO labeled BMSC-Luc/eGFP as compared to grafted unlabeled BMSC-Luc/eGFP, both at week 1 and week 2 post-implantation (Figure 6). At both time-points, results demonstrate a 2.9 ratio of in vivo BLI signal amplification due to GB MPIO labeling of BMSC-Luc/eGFP (for week 1 pO.OO36 and for week 2 pO.OO97).

Conclusion

These results confirm the above described in vitro observations. In addition, no significant difference was observed in BLI signal intensity between week I and week 2 post-injection, both for unlabeled (p = 0.81) and GB MPIO labeled (p = 0.66) BMSC-Luc/eGFP (Figure 6).

Example 4 In vivo enhancer effect in transplanted neural stem cells.

To further evaluate the effect of the beads, and using the same protocols and cells as in example 3 hereinbefore, beads with different fluorophores were used for incubation with the BMSC-Luc/eGFP. Next to glacial blue labeled beads mentioned before we include a dragon green labeled bead (cat n° MEO4F/9255, Bangs Laboratories, Fishers, IN, USA). This bead has a higher emission spectrum and is therefore unsuitable for BRET. We also included a bead labeled with Flash red (cat n° MEO4F/9486, Bangs Laboratories, Fishers, IN, USA), this has a lower emission spectrum and has a potential to be activated by the photons from luciferase, however it is probably too far away from the luciferase spectrum to be involved in BRET. As is shown in figure 7, the beads all showed an increased signal (p values all <0.01) on the bioluminescence measurement which was independent of the type of beads used.

References 1. Jansson,K., Bengtsson,L., Swedenborg,J., & Haegerstrand,A. In vitro endothelialization of bioprosthetic heart valves provides a cell monolayer with proliferative capacities and resistance to pulsatile flow. J. Thorac. Cardiovasc. Surg. 121, 108-115 (2001).

2. Bulte,J.W., Duncan,I.D., & Frank,J.A. In vivo magnetic resonance tracking of magnetically labeled cells after transplantation. I Cereb. Blood Flow Meta.h 22, 899-907 (2002).

3. Modo,M., Hoehn,M., & Bulte,J.W. Cellular MR imaging. Mo!. Imaging 4, 143-164 (2005).

4. Politi,L.S. MR-based imaging of neural stem cells. Neuroradiology 49, 523-534 (2007).

5. Bulte,J.W. & Kraitchmnan,D.L. Monitoring cell therapy using iron oxide MR contrast agents.

Curr. Pharm. Biotechnol. 5, 567-584 (2004).

6. Bulte,J.W., Ben Hur,T., Miller,B.R., Mizrachi-Kol,R., Einstein,O., Reinhartz,E., Zywicke,H.A., Douglas,T., & Frank,J.A. MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain. Magn Reson. Med. 50, 201-205 (2003).

7. Hoehn,M., Wiedermann,D., Justicia,C., Ramos-Cabrer,P., Kruttwig,K., Farr,T., & Himmelreich,U. Cell tracking using magnetic resonance imaging. J. Physiol 584, 25-30 (2007).

8. Chiancone,E., Ceci,P., Ilari,A., Ribacchi,F., & Stefanini,S. Iron and proteins for iron storage and detoxification. Biometals 17, 197-202 (2004).

9. Goldhawk,D.E., Lemaire,C., McCreary,C.R., McGirr,R., Dhanvantari,S., Thompson,R.T., Figueredo,R., Koropatnick,J., Foster,P., & Prato,F.S. Magnetic resonance imaging of cells overexpressing MagA, an endogenous contrast agent for live cell imaging. Mol. Imaging 8, 129- 139 (2009).

10. Vreys,R., Vande,V.G., Krylychkina,O., Vellema,M., Verhoye,M., Tirnmermans,J.P., Baekelandt,V., & Van der,L.A. MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain: validation of various MPIO labeling strategies. Neuroimage. 49, 2094-2103 (2010).

11. Kraitchrnan,D.L., Heldman,A.W., Atalar,E., Amado,L.C., Martin,B.J., Pittenger,M.F., Hare,J.M., & Bulte,J.W. In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation 107, 2290-2293 (2003).

12. Mills,P.H. & Ahrens,E.T. Theoretical MRI contrast model for exogenous T2 agents. Magn Reson. Med. 57, 442-447 (2007).

13. Ronsyn,M.W., Daans,J., Spaepen,G., Chatterjee,S., Vermeulen,K., D'Haese,P., Van Tendeloo,V.F., Van Marck,E., Ysebaert,D., Berneman,Z.N., Jorens,P.G., & Ponsaerts,P.

Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord. BMC. Biotechnol. 7, 90 (2007).

14. Virostko,J., Chen,Z., Fowler,M., Poffenberger,G., Powers,A.C., & Jansen,E.D. Factors influencing quantification of in vivo bioluminescence imaging: application to assessment of pancreatic islet transplants. Mol. Imaging 3, 333-342 (2004).

15. Tolar,J., Osborn,M., Bell,S., McElmurry,R., Xia,L., Riddle,M., Panoskaltsis-Mortari,A., Jiang,Y., Mclvor,R.S., Contag,C.H., Yant,S.R., Kay,M.A., Verfaillie,C.M., & Blazar,B.R. Real-time in vivo imaging of stem cells following transgenesis by transposition. Mol. Ther. 12, 42-48 (2005).

16. Min,J.J., Ahn,Y., Moon,S., Kim,Y.S., Park,J.E., Kim,S.M., Le,U.N., Wu,J.C., Joo,S.Y., Hong,M.H., Yang,D.H., Jeong,M.H., Song,C.H., Jeong,Y.H., Yoo,K.Y., Kang,K.S., & Bom,H.S.

In vivo bioluminescence imaging of cord blood derived mesenchymal stem cell transplantation into rat myocardium. Ann. Nuci. Med. 20, 165-170 (2006).

17. Swijnenburg,R.J., Schrepfer, S., Cao,F., Pearl,J.I., Xie,X., Connolly,A.J., Robbins,R.C., & Wu,J.C. In vivo imaging of embryonic stem cells reveals patterns of survival and immune rejection following transplantation. Ste,n Cells Dcv. 17, 1023-1029 (2008).

18. Giuriato,S., Faumont,N., Bousquet,E., Foisseau,M., Bibonne,A., Moreau,M., Al Saati,T., Felsher,D.W., Delsol,G., & Meggetto,F. Development of a conditional bioluminescent transplant model for TPM3-ALK-induced tumorigenesis as a tool to validate ALK-dependent cancer targeted therapy. CancerBiol. Ther. 6, 1318-1323 (2007).

19. Bergwerf,I., De Voclit,N., Tambuyzer,B., Verschueren,J., Reekmans,K., Daans,J., Ibrahiini,A., Van,T., V, Cliatterjee,S., Goossens,H., Jorens,P.G., Baekelandt,V., Ysebaert,D., Van Marck,E., Berneman,Z.N., Linden,A.V., & Ponsaerts,P. Reporter gene-expressing hone marrow-derived strornal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice. BMC. Biotechnol. 9, 1 (2009).

20. So,M.K., Xu,C., Loening,A.M., Gambhir,S.S., & Rao,J. Self-illuminating quantum dot conjugates for in vivo imaging. Nat. Biotechnol. 24, 339-343 (2006).

21. Hwang,d.W., Ko,H.Y., Kim,S.K., Kim,D., Lee,D.S., & Kim,S. Development of a quadruple imaging modality by using nanoparticles. Chemistry. 15, 93 87-9393 (2009).

22. Hargrave,P., Nicholson,P.W., Delpy,D.T., & Firbank,M. Optical properties of multicellular tumour spheroids. Phys. 1%ied. Biol. 41, 1067-1072 (1996).

Claims (44)

  1. What is claimed is: 1. Use of light-deflective particles, in the amplification or enhancement of a luminescent signal within a high scattering medium.
  2. 2. Use as claimed in claim 1, wherein said luminescent signal is a bioluminescent signal.
  3. 3. Use as claimed in claim 2, wherein said bioluminescent signal is created by exogenous and endogenous sources; in particular exogenous sources selected from transgenic expression of fluorescent or bioluminescent reporter genes; dyes; antibodies; nanobodies; or nanoparticles.
  4. 4. Use as claimed in any one of claims 1 to 3, wherein the light-deflective particles have light deflecting surface areas of from between and about 3x 1 0b0 mm2 to 0.25 mm2; in particular from between and about 3x10'° mm2 to 0.1 mm2; more in particular from between and about 3x101° mm2 to 1x103 mm2.
  5. 5. Use as claimed in any one of claims 1 to 3, wherein the light-deflective particles have a length of from about 30 nm to 400 microns (0.00003 -0.4 mm), a width of from about 10 nm to 400 microns (0. 0000 1-0.4 mm) and a thickness of 10 nm to 20 microns (0.0000 1 -0.02 mm).
  6. 6. Use as claimed in any one of claims 1 to 3, wherein the light-deflective particles are microspheres with a diameter of between and about 10 nm to 200 microns (0.0000 1 -0.2 mm), in particular with a diameter of between and about 10 nm to 20 microns (0.0000 1 -0.02 mm), more in particular with a diameter of between and about 10 nm to 200 nm (0.0000 1 -0. 0002 mm).
  7. 7. Use as claimed in any one of claims 1 to 3, wherein the light deflective particles comprise an outer membrane and an inner core.
  8. 8. Use as claimed in claim 7, wherein the outer membrane is made from an inert bio-compatible material, such as for example glass, ceramics, bio-compatible metals, bio-compatible polymers, or biodegradable materials.
  9. 9. Use as claimed in claim 8, wherein the bio-compatible polymers are selected from the group consisting of polystyrene, polyacrylic acid, silicone, polypropylene, polyester, polytetrafluoroethylene, polyethylene terephthalate, polyurethane, polymethylmethacrylate, and polymers of ethylenediamine, diethylenetriamine, allylamine or hydroxyethylmethacrylate.
  10. 10. Use as claimed in claim 8, wherein the biodegradable materials are selected from the group consisting of liposomes, biodegradable polymers and biodegradable polysaccharides.
  11. 11. Use as claimed in any one of claims 7 to 9 where the outher membrane is coated with substances such as but not limited to dextran, citrate, dimercaptosuccinic acid.
  12. 12. Use as claimed in any one of claims 7 to 10 where the outher membrane is functionalised by protein or peptide coating
  13. 13. Use as claimed in claim 12, wherein the proteins or peptides are antibodies or nanobodies.
  14. 14. Use as claimed in claim 12, wherein the proteins or peptides are ligands to membrane bound moieties such as but not limited to proteins, glycoproteins, phospholipids or any fraction thereof
  15. 15. Use as claimed in claim 12, wherein the outer membrane is functionalised with fluorescent proteins
  16. 16. Use as claimed in claim 12, wherein the outer membrane is functionalised with light emmiting proteins such as but not limited to firefly or renilla luciferase.
  17. 17. Use as claimed in claim 16, where only the fluorescent or light emmiting domain of the relevant proteins is used.
  18. 18. Use as claimed in Claim 7 where the inner core is made from the same material as the outer membrane or is at least partially consists of a low light scattering medium such as an optically translucent solution, solid, or gas.
  19. 19. Use as claimed in claim 18, wherein the optically translucent solution is selected from the group but not limited to water, saline buffers, optical gels, glass.
  20. 20. Use as claimed in claim 19, where the optically translucent solution is combined with fluorescent compounds such as but not limited to fluorescein isothiocyanate, rhodamine, coumarin, sulforhodamine 101 acid chloride, ethidium bromide, propidium iodide or 4',6-diamidino-2-phenylindole.
  21. 21. Use as claimed in claims 19 or 20, where the optical translucent solution is combined with ion indicators such as but not limited to the Ca++ dependent dyes FURA-2, FURA-2AM or INDO-1.
  22. 22. Use as claimed in any one of claims 19 to 21, where the optical translucent solution is combined with magnetic indicators for MRI.
  23. 23. Light-deflective particles, for use in the amplification or enhancement of a luminescent signal within a high scattering medium, said particles comprising an outer membrane and an inner core, and being characterized in that the inner core at least partially consists of a low light scattering medium such as an optically translucent solution or a gas.
  24. 24. The light-deflective particles according to claim 23, wherein the optically translucent solution is selected from the group but not limited to water, saline buffers, optical gels, glass.
  25. 25. The light-deflective particles according to any one of claims 23 to 24, where the optically translucent solution is combined with fluorescent compounds such as but not limited to fluorescein isothiocyanate, rhodamine, coumarin, sulforhodamine 101 acid chloride, ethidium bromide, propidium iodide or 4',6-diamidino-2-phenylindole.
  26. 26. The light-deflective particles according to any one of claims 23 to 25, where the optical translucent solution is combined with ion indicators such as but not limited to the Ca++ dependent dyes FURA-2, FURA-2AM or INDO-1.
  27. 27. The light-deflective particles according to any one of claims 23 to 26, where the optical translucent solution is combined with magnetic indicators for MRI.
  28. 28. The light-deflective particles according to any one of claims 23 to 27, wherein the outer mebrane is made from an inert bio-compatible material, such as for example glass, ceramics, bio-compatible metals, bio-compatible polymers, or biodegradable materials; in particular biodegradable materials.
  29. 29. The light-deflective particles according to claim 28, wherein the outer mebrane is made from an inert biodegradable material selected from the group consisting of poly(alkylene glycol), poly(2-hydroxyethyl methacrylate), poly(3 -hydroxypropyl methacrylamide), hydroxylated poly(vinyl pyrrolidone), sulfonated dextran, sulfonated polystyrene, fibrin, fibrinogen, cellulose, starch, collagen, hyaluronic acid, heparin, a graft copolymer of poly(L-lysine)-graft-co-poly(ethylene glycol), poly(L-lactide), poly(D,L-lactide), poly(glycolide), poly(L-lactide-co-glycolide), poly(D,L-lactide-co-glycolide), poly(caprolactone), poly(L-lactide-co-caprolactone), poly(D,L-lactide-co- caprolactone), polyhydroxyalkanoates, poly(3 -hydroxybutyrate), poly(4-hydroxybutyrate), poly(hydroxyvalerate), poly(3 -hydroxybutyrate-co-valerate), poly(4-hydroxybutyrate-co-valerate), poly(ester amides), poly(anhydrides), poly(carbonates), poly(trimethylene carbonate-co-glycolide), poly(trimethylene carbonate-co-L-lactide), poly(trimethylene carbonate-co-D,L-lactide), poly(dioxanone), poly(phosphazenes), poly(orthoesters), poly(tyrosine-co-carbonates), polyalkylene oxalates, poly(glycerol-co-sebacic acid esters), cyanoacrylates, poly(amino acids), poly(lysine), poly(glutamic acid) and mixtures thereof.
  30. 30. The light-deflective particles according to any one of claims 23 to 29, where the outher membrane is coated with substances such as but not limited to dextran, citrate, dimercaptosuccinic acid.
  31. 31. The light-deflective particles according to any one of claims 23 to 29, where the outher membrane is functionalised by protein or peptide coating
  32. 32. The light-deflective particles according to claim 31, wherein the proteins or peptides are antibodies or nanobodies.
  33. 33. The light-deflective particles according to claim 31, wherein the proteins or peptides are ligands to membrane bound moieties such as but not limited to proteins, glycoproteins, phospholipids or any fraction thereof.
  34. 34. The light-deflective particles according to any one of claims 23 to 29, wherein the outer membrane is functionalised with fluorescent proteins
  35. 35. The light-deflective particles according to any one of claims 23 to 29, wherein the outer membrane is functionalised with light emmiting proteins such as but not limited to firefly or renilla luciferase
  36. 36. The light-deflective particles according to claim 35, where only the fluorescent or light emmiting domain of the relevant proteins is used.
  37. 37. A method of amplifying or enhancing a luminescent signal within a high scattering medium, said method comprising adding a luminescent enhacing amount of deflective particles as defined in any one of claims 4 to 22, to said high scattering medium.
  38. 38. The method as claimed in claim 37, wherein said luminescent signal is a bioluminescent signal.
  39. 39. The method as claimed in claim 38, wherein said bioluminescent signal is created by exogenous and endogenous sources; in particular exogenous sources selected from transgenic expression of fluorescent or bioluminescent reporter genes; dyes; antibodies; nanobodies; or nanoparticles.
  40. 40. The method as claimed in claim 38, wherein said bioluminescent signal is used for diagnostic in vivo imaging; and the method comprises administering to a subject a luminescent enhacing amount of deflective particles as defined in any one of claims 4 to 22.
  41. 41. The method as claimed in claim 38, wherein said bioluminescent signal is used for diagnostic in vitro imaging; and the method comprises administering to an in vitro assay a luminescent enhacing amount of deflective particles as defined in any one of claims 4 to 22.
  42. 42. The method as claimed in claim 37, wherein said luminescent signal is used for monitoring bioreactors; and the method comprises administering to a bioreactor a luminescent enhacing amount of deflective particles as defined in any one of claims 4 to 22.
  43. 43. The method as claimed in claim 37, wherein said luminescent signal is used for phototherapy; and the method comprises administering to a subject in need thereof a luminescent enhacing amount of deflective particles as defined in any one of claims 4 to 22.
  44. 44. The method as claimed in claim 37, wherein said luminescent signal is used for optogenetics; and the method comprises administering to a subject in need thereof a luminescent enhacing amount of deflective particles as defined in any one of claims 4 to 22.
GB201008768A 2010-05-26 2010-05-26 Luminescence enhancing beads for bio-imaging Withdrawn GB2480640A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB201008768A GB2480640A (en) 2010-05-26 2010-05-26 Luminescence enhancing beads for bio-imaging

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
GB201008768A GB2480640A (en) 2010-05-26 2010-05-26 Luminescence enhancing beads for bio-imaging

Publications (2)

Publication Number Publication Date
GB201008768D0 GB201008768D0 (en) 2010-07-14
GB2480640A true GB2480640A (en) 2011-11-30

Family

ID=42371002

Family Applications (1)

Application Number Title Priority Date Filing Date
GB201008768A Withdrawn GB2480640A (en) 2010-05-26 2010-05-26 Luminescence enhancing beads for bio-imaging

Country Status (1)

Country Link
GB (1) GB2480640A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105961049A (en) * 2016-05-25 2016-09-28 河北大学 Optomagnetic processing device for biological ultra-weak luminescence research

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0876606A1 (en) * 1995-09-28 1998-11-11 Georgia Tech Research Corporation Particle enhanced spectroscopic detection
EP1051607A1 (en) * 1998-01-21 2000-11-15 Bayer Corporation Optical sensors with reflective materials and methods for producing such optical sensors
EP1261857A1 (en) * 2000-03-06 2002-12-04 The Johns Hopkins University Scatter controlled emission for optical taggants and chemical sensors
GB2446019A (en) * 2007-01-27 2008-07-30 Univ Cranfield The enhancement of light penetration in tissues using chemical agents and ultrasound

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0876606A1 (en) * 1995-09-28 1998-11-11 Georgia Tech Research Corporation Particle enhanced spectroscopic detection
EP1051607A1 (en) * 1998-01-21 2000-11-15 Bayer Corporation Optical sensors with reflective materials and methods for producing such optical sensors
EP1261857A1 (en) * 2000-03-06 2002-12-04 The Johns Hopkins University Scatter controlled emission for optical taggants and chemical sensors
GB2446019A (en) * 2007-01-27 2008-07-30 Univ Cranfield The enhancement of light penetration in tissues using chemical agents and ultrasound

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105961049A (en) * 2016-05-25 2016-09-28 河北大学 Optomagnetic processing device for biological ultra-weak luminescence research
CN105961049B (en) * 2016-05-25 2019-01-01 河北大学 A kind of optomagnetic processing unit of Ultra-weak Bioluminescence research

Also Published As

Publication number Publication date
GB201008768D0 (en) 2010-07-14

Similar Documents

Publication Publication Date Title
Frank et al. Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents
Smith et al. Quantum Dot Nanocrystals for In Vivo Molecular and Cellular Imaging¶
Wang et al. Immunolabeling and NIR-excited fluorescent imaging of HeLa cells by using NaYF4: Yb, Er upconversion nanoparticles
Zhou et al. Upconversion nanophosphors for small-animal imaging
Smith et al. Engineering luminescent quantum dots for in vivo molecular and cellular imaging
Arbab et al. Cellular magnetic resonance imaging: current status and future prospects
Markides et al. Biocompatibility and toxicity of magnetic nanoparticles in regenerative medicine
Byers et al. Quantum dots brighten biological imaging
Xu et al. Complex heterogeneous tissue constructs containing multiple cell types prepared by inkjet printing technology
Souza et al. Three-dimensional tissue culture based on magnetic cell levitation
Chan et al. MRI-detectable pH nanosensors incorporated into hydrogels for in vivo sensing of transplanted-cell viability
Walczak et al. Applicability and limitations of MR tracking of neural stem cells with asymmetric cell division and rapid turnover: the case of the shiverer dysmyelinated mouse brain
Lyons Advances in imaging mouse tumour models in vivo
Hemmer et al. Upconverting and NIR emitting rare earth based nanostructures for NIR-bioimaging
Shi et al. Fluorescent polystyrene–Fe3O4 composite nanospheres for in vivo imaging and hyperthermia
Jaffer et al. Seeing within: molecular imaging of the cardiovascular system
Delcroix et al. Mesenchymal and neural stem cells labeled with HEDP-coated SPIO nanoparticles: in vitro characterization and migration potential in rat brain
Cheng et al. Multifunctional upconversion nanoparticles for dual‐modal imaging‐guided stem cell therapy under remote magnetic control
Xie et al. Three-dimensional cell-scaffold constructs promote efficient gene transfection: implications for cell-based gene therapy
Zako et al. Cyclic RGD peptide-labeled upconversion nanophosphors for tumor cell-targeted imaging
Bhirde et al. Nanoparticles for cell labeling
Wang et al. Rapid movement of microtubules in axons
Xing et al. Quantum dot bioconjugates for in vitro diagnostics & in vivo imaging
Ferreira et al. New opportunities: the use of nanotechnologies to manipulate and track stem cells
Altınogˇlu et al. Near-infrared emitting fluorophore-doped calcium phosphate nanoparticles for in vivo imaging of human breast cancer

Legal Events

Date Code Title Description
WAP Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1)