WO2005017539A2 - Imaging pathology - Google Patents

Abstract

A method for differentiating a diseased tissue from normal host tissue in subject by administering a bifunctional probe which comprises an optical imaging moiety and a magnetic resonance moiety. This method can provide delineation of pathology margins by pre-operative magnetic resonance imaging and/or by intra-operative optical imaging. The probes can therefore be used to differentiate diseased tissues from normal tissues during surgery.

Description

IMAGING PATHOLOGY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application Serial No. 60/495,006, filed on August 14, 2003, the entire contents of which is incorporated herein by reference. TECHNICAL FIELD This invention relates generally to bifunctional magnetic resonance/optical probes.

BACKGROUND The determination of tumor margins can be crucial to successful outcomes for brain tumor patients undergoing surgery. However, the correspondence between a pre-operative image of a pathology, e.g., a tumor or lesion, and an intra-operative visual presentation of the pathology can be complicated, for example, by changes in projection between the pre-operative image and visual presentation during surgery and shifts in position of the pathology caused by surgical intervention. For example, in brain surgery, the release of pressure caused by the tumor during surgery can change the position and/or volume of the tumor.

SUMMARY This invention relates generally to bifunctional magnetic resonance/optical probes. In one aspect, the invention relates to methods for differentiating a diseased tissue from normal host tissue in a subject by (a) administering a bifunctional probe to the subject, wherein the probe includes an optical imaging moiety and a magnetic resonance imaging moiety; (b) obtaining a preoperative magnetic resonance image of the diseased tissue and the host tissue, wherein the portion of the magnetic resonance image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins of the diseased tissue; (c) removing or displacing one or more layers of cutaneous matter, subcutaneous matter, skeletal matter, vascular tissue or other host tissue, thereby partially or fully exposing the diseased tissue and host tissue; and (d) obtaining at least one optical image of the exposed diseased tissue and host tissue during and/or after step (c), wherein for each optical image obtained, the portion of the image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins or changes in the margins of the diseased tissue in response to step (c). In another aspect, this invention relates to methods for differentiating a diseased tissue (e.g., a pathology, e.g., a tumor, a lesion, or an abscess) from normal host tissue in a subject under intra-operative conditions, the methods for removing or exploring the diseased tissue, the methods include: (a) administering to the subject a probe comprising an optical imaging moiety, wherein the probe is preferentially sequestered into one or more host response cells in the diseased tissue or at its periphery; (b) removing or displacing one or more layers of cutaneous matter, subcutaneous matter, skeletal matter, vascular tissue or host tissue, thereby partially or fully exposing the diseased tissue and host tissue; and (c) obtaining at least one optical image of the exposed diseased tissue and host tissue during and/or after step (b), wherein for each optical image obtained, the portion of the image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins or changes in the margins of the diseased tissue in response to step (b). In a further aspect, the invention relates to a computer-readable medium on which is recorded one or more magnetic resonance images and/or one or more optical images that differentiate a diseased tissue from normal host tissue in a subject, wherein the optical image is generated by a process that includes any of the methods described herein. In another aspect, this invention relates to the use of a bifunctional probe including an optical imaging moiety and a magnetic resonance imaging moiety to differentiate a diseased tissue from normal host tissue in a subject under intra- operative conditions to remove or explore the diseased tissue. The invention also relates to the use of a bifunctional probe including an optical imaging moiety and a magnetic resonance imaging moiety in the manufacture of a medicament to differentiate a diseased tissue from normal host tissue in a subject under intra-operative conditions to remove or explore the diseased tissue. Embodiments can include one or more of the following features. The methods can further include obtaining a preoperative magnetic resonance image of the diseased tissue and host tissue after step (a) and before step (b), wherein the portion of the magnetic resonance image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins of the diseased tissue. The methods can further include (d) resecting the diseased tissue; and (e) obtaining at least one optical image of the exposed diseased tissue and host tissue during step (d), wherein for each optical image obtained, the portion of the image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins or changes in the margins of the diseased tissue in response to step (d). The host tissue can be a brain tissue, breast tissue, a lymph node (e.g., a sentential lymph node), or gastrointestinal tissue. The diseased tissue can be a tumor (e.g., a tumor associated with the brain, lung, breast, or colon). The probe can have a half-life in the blood pool of at least about 2 hours (e.g., at least about 6 hours, at least about 12 hours, at least about 20 hours, at least about 30 hours, or at least about 40 hours. The magnetic resonance imaging moiety can include a magnetic nanoparticle (e.g., a magnetic metal oxide, e.g., a superparamagnetic metal oxide). The metal oxide can be iron oxide. In certain embodiments, the nanoparticle can be an amino-derivatized cross- linked iron oxide nanoparticle, and can have an average diameter of from about 5 nm to about 100 nm (e.g., from about 20 nm to about 80 nm, from about 40 nm to about 60 nm). In various embodiments, the optical imaging moiety can include at least one fluorochrome. The fluorochrome can be a near infrared fluorochrome (e.g., Cy5.5, Cy5, Cy 7, Alexa Fluoro 680, Alexa Fluoro 750, IRD41, IRD700, NIR-1, LaJolla Blue, indocyanine green (ICG), indotricarbocyanine (ITC), or a chelated lanthanide compound). In certain embodiments, the near infrared fluorochrome can be Cy5.5. The optical imaging moiety and the magnetic resonance imaging moiety can be covalently linked (e.g., the optical imaging moiety and the magnetic resonance imaging moiety can be covalently linked by a bond between a carbonyl group on the optical imaging moiety and an amino group on the magnetic resonance imaging moiety). The probe can further include a polymeric backbone, in which each of the optical imaging moiety and the magnetic resonance imaging moiety can be linked to the polymeric backbone. The optical imaging moiety and the magnetic resonance imaging moiety can be covalently linked to the backbone. The magnetic resonance imaging moiety can include a chelator moiety and a chelated paramagnetic or superparamagnetic metal atom or ion. The ion can be Gd . The chelator can be selected from 1, 4,7,10-tetraazacyclodo-decane-N,N',N",N"'- tetraacetic acid; l,4,7,10-tetraaza-cyclododecane-N,N',N"-triacetic acid; 1,4,7- tris(carboxymethyl)- 10-(2'-hydroxypropyl)- 1 ,4,7, 10-tetraazocyclodecane; 1 ,4,7- triazacyclonane-N,N',N"-triacetic acid; 1,4,8,11-tetraazacyclotetra-decane- N,N',N",N'"-tetraacetic acid; diethylenetriamine-pentaacetic acid (DTP A); ethylenedicysteine; bis(aminoethanethiol)carboxylic acid; triethylenetetraamine- hexaacetic acid; ethylenediamine-tetraacetic acid (EDTA); 1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid; N-(hydroxy-ethyl)ethylenediaminetriacetic acid; nitrilotriacetic acid; and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid. The polymeric backbone can include a polysaccharide, a nucleic acid, polypeptide or a synthetic polymer. The polymeric backbone can further include at least one protective chain covalently linked to the backbone. The protective chain can be polyethylene glycol, methoxypolyethylene glycol, methoxypolypropylene glycol, copolymers of polyethylene glycol and methoxypolypropylene glycol, dextran, or polylactic-polyglycolic acid. The diseased tissue can be associated with or cause changes in one or more microglial cells. In some embodiments, substantially all of the probe is sequestered by the microglial cells, and the portion of the image associated with the host tissue can represent a tissue that is substantially free of the diseased tissue. The probe can further include a GABA receptor. The diseased tissue margins delineated in the magnetic resonance image can be substantially congruent with the diseased tissue margins delineated in at least one of the optical images obtained in step (c). The subject can be a mammal (e.g., a mouse, rat, cow, sheep, pig, rabbit, goat, horse, monkey, dog, cat, or human). In certain embodiments, the subject can be a human. Embodiments can have one or more of the following advantages. In certain embodiments, the probes can have a relatively long duration for detection, for example, relative to extracellular perfusion based agents. In certain embodiments, optical imaging during surgery can be performed both pre-operatively (e.g., organism intact) or at various levels of dissection. For example, as skin and layers of tissue are removed, or as blood vessels are clamped or pushed aside, repeated optical images can still be made. In certain embodiments, intra-operative optical imaging of a pathology can enhance the ease of obtaining an image during surgery, thereby reducing the complexity and cost of obtaining an image during surgery relative to, for example, obtaining an intra-operative MR imaging of the same pathology and host tissue. In certain embodiments, optical imaging can be performed using relatively short acquisition times (e.g., about 500 msec), and the use of near infrared fluorescence can minimize the likelihood of tissue auto-fluorescence. In certain embodiments, the use of near infrared fluorescence can provide visualization of the probes through overlaying tissue (e.g., > 1 millimeter (mm), e.g., before complete dissection of the tumor is accomplished). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

DESCRIPTION OF DRAWINGS FIG 1 A is a proton density- weighted MR image of a tumor bearing rat brain after administration of Cy5.5-CLIO. FIG. IB is a T2 weighted MR image of the tumor bearing rat brain of FIG. 1A after administration of Cy5.5-CLIO. FIG. 1C is an image that shows the margins of the tumor bearing rat brain of FIGS. 1A and IB as determined by Hematoxylin-Eosin staining. The image was obtained after histological processing of the rat brain. FIG.D is an amplified Prussian blue stained image of the tumor bearing rat brain of FIGS. 1 A and IB. The image was obtained after histological processing of the rat brain and demonstrates the accumulation of iron in the tumor. FIG. 2A is an image (post-craniotomy) of a rat bearing a 3 millimeter (mm) 9L glioma tumor in the white light channel. The image was obtained after administration of Cy5.5-CLIO. FIG. 2B is an image (post-craniotomy) of the rat of FIG. 2 A in the green fluorescence protein (GFP) channel. The image was obtained after administration of Cy5.5-CLIO. FIG. 2C is an image (post-craniotomy) of the rat of FIG. 2 A in the Cy5.5 channel. The image was obtained after administration of Cy5.5-CLIO. FIG. 2D is an image that shows the margins of the tumor delineated in FIGS. 2A-2C as determined by Hematoxylin-Eosin staining. The image was obtained after histological processing of the rat brain. FIG. 2E is an image of the tumor delineated in FIGS. 2A-2C in the GFP channel. The image was obtained after histological processing of the rat brain. FIG. 2F is an image of the tumor delineated in FIGS. 2A-2C in Cy5.5 channel. The image was obtained after histological processing of the rat brain. FIG. 3 is a graphical representation of Cy5.5 positive areas and GFP positive areas, demonstrating the accuracy of Cy5.5 fluorescence in determining true tumor extent as defined by gold standard GFP fluorescence. Five brain tumors were sliced in 4-8 slices each (25 slices total) and Cy5.5 and GFP positive areas on slices were determined by region of interest analysis. FIG. 4 A is an image of cells from the center of the tumor delineated in FIGS. 2A-2C as determined by laser-scanning confocal microscopy (original magnification x 200). The image shows Cy5.5-CLIO distribution in the Cy5.5 channel. FIG. 4B is an image of cells from the center of the tumor delineated in FIGS. 2A-2C as determined by laser-scanning confocal microscopy (original magnification x 200) in the GFP channel. FIG. 4C is an image of cells from the center of the tumor delineated in FIGS. 2A-2C as determined by laser-scanning confocal microscopy (original magnification x 200). The image is an anti-CD 1 lb stain for microglia and macrophages in the rhodamine channel. FIG. 4D is an overlay of the image shown in FIG. 4A and the image shown in FIG. 4C. FIG. 4E is an overlay of the image shown in FIG. 4A and the image shown in

FIG. 4B. FIG. 4F is an image of cells from the tumor (delineated in FIGS. 2A-2C)-brain interface as determined by laser-scanning confocal microscopy (original magnification x 10). The image shows the tumor border in the GFP channel. FIG. 4G is an image of cells from the tumor (delineated in FIGS. 2A-2C)- brain interface as determined by laser-scanning confocal microscopy (original magnification x 10). The image shows the tumor border as delineated by anti-CDl lb staining for microglia and macrophages in the rhodamine channel. FIG. 4H is an overlay of the image shown in FIG. 4F and the image shown in FIG. 4G. Cells positive for CD1 lb extend slightly beyond the border of GFP fluorescence. FIG. 5 is a graphical representation of relative number of U937 cells vs. fluorescence signal intensity revealing cellular uptake of Cy5.5-CLIO in culture for this cell line. "Control" cells are the same cell line but were not exposed to Cy5.5- CLIO. Cells exposed to Cy5.5-CLIO at a concentration of 100 mgFe/ml demonstrate uptake reported as increased fluorescence compared to control cells. Culture with phorbol 12-myristate 13-acetate (PMA) for 7 days. PMA was replaced every 2 days. Cy5.5-CLIO incubation: 20 hours. FIG. 6 is a graphical representation of relative number of C6 glioma cells vs. fluorescence signal intensity revealing cellular uptake of Cy5.5-CLIO in culture for this cell line. "Control" cells are the same cell line, but were not exposed to Cy5.5- CLIO. Cells exposed to Cy5.5-CLIO at a concentration of 100 mgFe/ml demonstrate uptake reported as increased fluorescence compared to control cells. Cy5.5-CLIO incubation: 24 hours. FIG. 7 is a graphical representation of relative number of Gli36 glioma cells vs. fluorescence signal intensity revealing cellular uptake of Cy5.5-CLIO in culture for this cell line. "Control" cells are the same cell line, but were not exposed to Cy5.5-CLIO. Cells exposed to Cy5.5-CLIO at a concentration of 100 mgFe/ml demonstrate uptake reported as increased fluorescence compared to control cells. Cy5.5-CLIO incubation: 24 hours. FIG. 8 is a graphical representation of relative number of LLC (Lewis lung carcinoma) cells vs. fluorescence signal intensity revealing cellular uptake of Cy5.5- CLIO in culture for this cell line. "Control" cells are the same cell line, but were not exposed to Cy5.5-CLIO. Cells exposed to Cy5.5-CLIO at a concentration of 100 mgFe/ml demonstrate uptake reported as increased fluorescence compared to control cells. Cy5.5-CLIO incubation: 24 hours. FIG. 9 is a graphical representation of relative number of HT29 colon cancer cells vs. fluorescence signal intensity revealing cellular uptake of Cy5.5-CLIO in culture for this cell line. "Control" cells are the same cell line, but were not exposed to Cy5.5-CLIO. Cells exposed to Cy5.5-CLIO at a concentration of 100 mgFe/ml demonstrate uptake reported as increased fluorescence compared to control cells. Cy5.5-CLIO incubation: 24 hours. FIG. 10A is an image (post-craniotomy) of a mouse bearing a GH36 brain tumor in the white light channel. The image was obtained after administration of Cy5.5-CLIO and demonstrates the appearance of the brain surface. FIG. 10B is an image (post-craniotomy) of the mouse of FIG. 10A in the green fluorescence protein (GFP) channel. The image was obtained after administration of Cy5.5-CLIO and demonstrates near infrared (NIR) fluorescence after probe injection. FIG. IOC is an image (post-craniotomy) of the mouse of FIG. 10A in the

Cy5.5 channel. The image was obtained after administration of Cy5.5-CLIO and demonstrates NIR fluorescence after probe injection. FIG. 10D is a normalized histogram of surface signal of GFP and Cy5.5 fluorescence, which demonstrates that improved tissue penetration at wavelengths in the far red and NIR compared to GFP allows the determination of tumor location even at a distance from the surface. The dark line in the histogram corresponds to Cy5.5, and the light line in the histogram corresponds to GFP. FIG. 10E is an image (post-craniotomy) of a mouse bearing a 9L brain tumor in the white light channel. The image was obtained after administration of Cy5.5- CLIO and demonstrates the appearance of the brain surface. FIG. 1 OF is an image (post-craniotomy) of the mouse of FIG. 10E in the green fluorescence protein (GFP) channel. The image was obtained after administration of Cy5.5-CLIO and demonstrates NTR fluorescence after probe injection. FIG. 10G is an image (post-craniotomy) of the mouse of FIG. 10E in the Cy5.5 channel. The image was obtained after administration of Cy5.5-CLIO and demonstrates NIR fluorescence after probe injection. FIG. 1 OH is a normalized histogram of surface signal of GFP and Cy5.5 fluorescence, which demonstrates that improved tissue penetration at wavelengths in the far red and NIR compared to GFP allows the determination of tumor location even at a distance from the surface. The dark line in the histogram corresponds to Cy5.5, and the light line in the histogram corresponds to GFP. FIG. 10J is an image (post-craniotomy) of a mouse bearing a 9L brain tumor in the white light channel. The image was obtained after administration of Cy5.5- CLIO and demonstrates the appearance of the brain surface. FIG. 1 OK is an image (post-craniotomy) of the mouse of FIG. 10J in the green fluorescence protein (GFP) channel. The image was obtained after administration of Cy5.5-CLIO and demonstrates NIR fluorescence after probe injection. FIG. 10L is an image (post-craniotomy) of the mouse of FIG. 10J in the Cy5.5 channel. The image was obtained after administration of Cy5.5-CLIO and demonstrates NIR fluorescence after probe injection.. FIG. 10M is a normalized histogram of surface signal of GFP and Cy5.5 fluorescence, which demonstrates that improved tissue penetration at wavelengths in the far red and NIR compared to GFP allows the determination of tumor location even at a distance from the surface. The dark line in the histogram corresponds to Cy5.5, and the light line in the histogram corresponds to GFP. FIG. 11A is a high magnification of Gli36 tumor border, which demonstrates relative area on a histological slide determined by Cy5.5 fluorescence representing area of probe uptake vs. area determined by GFP (representing "true" tumor area, since only the tumor cells used in this experiment express GFP). The high correlation suggests that the probe is distributed to a volume that highly approximates and minimally overestimates true tumor margins. FIG. 1 IB is a high magnification of 9L tumor border, which demonstrates relative area on a histological slide determined by Cy5.5 fluorescence representing area of probe uptake vs. area determined by GFP (representing "true" tumor area, since only the tumor cells used in this experiment express GFP). The high correlation suggests that the probe is distributed to a volume that highly approximates and minimally overestimates true tumor margins. FIG. 11C is a high magnification of C6 tumor border, which demonstrates relative area on a histological slide determined by Cy5.5 fluorescence representing area of probe uptake vs. area determined by GFP (representing "true" tumor area, since only the tumor cells used in this experiment express GFP). The high correlation suggests that the probe is distributed to a volume that highly approximates and minimally overestimates true tumor margins.

DETAILED DESCRIPTION This invention relates generally to bifunctional magnetic resonance/optical probes and related methods of use. The inventors have discovered that bifunctional probes having a magnetic resonance (MR) imaging moiety (e.g., superparamagnetic iron oxides and Gd- chelates, Mn-chelates) and an optical imaging moiety (e.g., a fluorochrome, e.g., a near infrared fluorochrome, e.g., Cy5.5) can provide relatively accurate delineation of pathology margins (e.g., tumor margins) by pre-operative magnetic resonance imaging and/or by intra-operative optical imaging. The probes can therefore be used to differentiate diseased tissues from normal host tissues (e.g., during surgery). While not wishing to be bound by theory, it is believed that pathology margin delineation is enhanced by sequestration and uptake of the probes into host response cells (e.g., activated microglia in the pathology or at its periphery) and/or into the cells of the pathology itself. In some embodiments, the probes can be preferentially sequestered into such host response cells (e.g., the probes are sequestered into host response cells at a rate that is faster relative to the rate of sequestration into normal host cells or the diseased tissue cells). In certain embodiments, bifunctional probes can be preferentially sequestered. Again, while not wishing to be bound by theory, it is belived that any probe that is preferentially sequestered by host response cells can provide enhanced margin delineation. It is also believed that the probes can manifest their locations in both pre-operative MR images and intra-operative optical images, because the probes will generally be localized in the same cells (e.g., microglia) in both the preoperative and intra-operative anatomies. As a result, the pathology margins delineated in an MR image can have a relatively high degree of congruency with the pathology margins delineated in an optical image. This correspondence between MR and optical images also provides a user with the flexibility to exploit only one of the probe's reporter capabilities (e.g., the optical reporting capability) if desired. Further, the uptake of the probes into, for example, peripheral microglia can provide a user with a visual lock on the pathology margins during surgery. For example, a user can track changes in pathology margins as a pathology is distorted (e.g., in volume or shape) during a surgical procedure (e.g., a tumor resection) by obtaining repeated (e.g., continuous) optical images of the intra-operative anatomy.

Bifunctional Probe Structure In general, the bifunctional probes include one or more magnetic resonance imaging moieties and one or more optical imaging moieties. In some embodiments, the probes include a magnetic resonance imaging moiety and an optical imaging moiety which can be linked to one another, for example, by one or more covalent bonds, by one or more covalent associations, or any combination thereof. In certain embodiments, the optical imaging moiety and the magnetic resonance imaging moiety are linked to one another by a covalent bond between a carbonyl carbon on the optical imaging moiety and a nitrogen atom on the magnetic resonance imaging moiety. In certain embodiments, such probes can be assembled by linking a precursor optical imaging moiety having, for example, an NHS ester, to a precursor magnetic resonance imaging moiety having a free amino group. In some embodiments, the magnetic resonance imaging moiety can include a magnetic nanoparticle, (e.g., magnetic metal oxide, such as superparamagnetic iron oxide). In certain embodiments, the magnetic nanoparticle can be a small paramagnetic iron oxide (SPIO) or an ultra-small paramagnetic iron oxide (USPIO). In certain embodiments, the magnetic nanoparticle can be a coated, cross-linked iron oxide (e.g., an iron oxide nanoparticle coated with aminated, cross-linked dextran, e.g., (CLIO)). The magnetic metal oxide can also comprise cobalt, magnesium, zinc, or mixtures of these metals with iron. The term "magnetic" as used herein means materials of high positive magnetic susceptibility such as superparamagnetic compounds and magnetite, gamma ferric oxide, or metallic iron. In certain embodiments, the magnetic nanoparticle can have a relatively high relaxivity, i.e., strong effect on water relaxation. In some embodiments, the magnetic nanoparticle (e.g., including the nanoparticle and a coating, e.g., a dextran coating) can have an average diameter of from about 5 nanometers (nm) to about 100 nm (e.g., from about 20 nm to about 80 nm, from about 40 nm to about 60 nm; e.g., 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45, nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm). In some embodiments, the magnetic resonance imaging moiety can include a chelator moiety and a chelated paramagnetic or superparamagnetic metal atom or ion. Various chelating moieties are known, and can be incorporated into a probe. In addition, novel chelating moieties can be discovered in the future and can be incorporated into a probe. In certain embodiments, the chelating moiety does not form a covalent bond with the paramagnetic or superparamagnetic metal or metal oxide. In some embodiments, the chelating moiety forms a thermodynamically and kinetically stable, non-covalent coordination complex or ionic complex with (iron) Fe³⁺, (gadolinium) Gd³⁺, (dysprosium) Dy^3-1", (europium) Eu³⁺, (manganese) Mn²⁺, or other useful metals (e.g., praseodymium (Pr), neodymium (Nd), samarium (Sm), terbium (Tb), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), or lutetium (Lu), or an ion or metal oxide thereof). In some embodiments, the chelator can be 1, 4,7,10-tetraazacyclodo-decane- N,N',N",N'"-tetraacetic acid; l,4,7,10-tetraaza-cyclododecane-N,N',N"-triacetic acid; 1 ,4,7-tris(carboxymethyl)- 10-(2'-hydroxypropyl)- 1 ,4,7,10-tetraazocyclodecane; 1 ,4,7- triazacyclonane-N,N',N"-triacetic acid; 1,4,8,11 -tetraazacyclotetra-decane- N,N',N",N'"-tetraacetic acid; diethylenetriamine-pentaacetic acid (DTP A); ethylenedicysteine; bis(aminoethanethiol)carboxylic acid; triethylenetetraamine- hexaacetic acid; ethylenediamine-tetraacetic acid (EDTA); 1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid; N-(hydroxy-ethyl)ethylenediaminetriacetic acid; nitrilotriacetic acid; and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid. The optical imaging moiety can be any moiety that interacts with light (e.g., a moiety that can emit detectable energy after excitation with light) and can include optically detectable agents, optically detectable dyes, optically detectable contrast agents, and/or optical dyes. In some embodiments, the optical imaging moiety can be a fluorescent moiety (e.g., a fluorochrome). In other embodiments, the optical imaging moiety can be a phosphorescent moiety. In some embodiments, the optical imaging moiety can be a near infrared fluorochrome (e.g., fluorochromes having excitation and emission wavelengths in the near infrared spectrum, e.g., 650-1300 nm). While not wishing to be bound by theory, it is believed that use of this portion of the electromagnetic spectrum can maximize tissue penetration and minimize absorption by physiologically abundant absorbers such as hemoglobin (< 650 nm) and water (>1200 nm). Various near infrared fluorochrome are commercially available and can be used to construct probes described herein. Exemplary fluorochromes include Cy5.5, Cy5 and Cy7 (Amersham, Arlington Hts., IL); IRD41 and IRD700 (LI-COR, Lincoln, NE); Alexa Fluor® 680, Alexa Fluor® 450 (Molecular Probes, Eugene, OR); R-1, (Dejindo, Kumamoto, Japan); LaJolla Blue (Diatron, Miami, FL); indocyanine green (ICG) and its analogs (Licha et al., 1996, SR7E 2927:192-198; Ito et al., U.S. Patent No. 5,968,479); indotricarbocyanine (ITC; WO 98/47538); and chelated lanthanide compounds. Fluorescent lanthanide metals include europium and terbium.

Fluorescence properties of lanthanides are described in Lackowicz, 1999, Principles of Fluorescence Spectroscopy, 2^nd Ed., Kluwar Academic, New York. In some embodiments, the optical imaging moiety can be porphrin, quantum dot, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methylcoumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade

Bleum™, or Texas Red. Other optical imaging moieties can be selected as desired, for example, from Molecular Probes. In some embodiments, the bifunctional probes can further include a polymeric backbone. Probe polymeric backbone design will depend on considerations such as biocompatibility (e.g., toxicity and immunogenicity), serum half-life, useful functional groups (e.g., for conjugating MR imaging moieties and optical imaging moieties), and cost. Useful types of polymeric backbones include polypeptides (polyamino acids), polyethyleneamines, polysaccharides, aminated polysaccharides, aminated oligosaccharides, polyamidoamines, polyacrylic acids, and polyalcohols. In some embodiments the backbone includes a polypeptide formed from L- amino acids, D-amino acids, or a combination thereof. Such a polypeptide can be, e.g., a polypeptide identical or similar to a naturally occurring protein such as albumin, a homopolymer such as polylysine, or a copolymer such as a D-tyr-D-lys copolymer. When lysine residues are present in the polymeric backbone, the e-amino groups on the side chains of the lysine residues can serve as convenient reactive groups for covalent linkage to MR imaging moieties and/or optical imaging moieties. When the polymeric backbone is a polypeptide, the molecular weight of the probe can be from about 2 kiloDaltons (kD) to about 1000 kD (e.g., from about 4 kD to about 500 kD). A polymeric backbone can be chosen or designed so as to have a suitably long in vivo persistence (e.g., half-life) inherently. In some embodiments, a rapidly- biodegradable polymeric backbone such as polylysine can be used in combination with covalently-linked protective chains. Examples of useful protective chains include polyethylene glycol (PEG), methoxypolyethylene glycol (MPEG), methoxypolypropylene glycol, polyethylene glycol-diacid, polyethylene glycol monoamine, MPEG monoamine, MPEG hydrazide, and MPEG imidazole. The protective chain can also be a block-copolymer of PEG and a different polymer such as a polypeptide, polysaccharide, polyamidoamine, polyethyleneamine or polynucleotide. Synthetic, biocompatible polymers are discussed generally in Holland et al., 1992, "Biodegradable Polymers," Advances in Pharmaceutical Sciences, 6:101-164. In certain embodiments, a polymeric backbone-protective chain combination can be methoxypoly(ethylene)glycol-succinyl-N-e-poly-L-lysyine (PL-MPEG). The synthesis of this material, and other polylysine backbones with protective chains, is described in Bogdanov et al., U.S. Patent No. 5,593,658 and Bogdanov et al., 1995, Advanced Drug Delivery Reviews, 16:335-348, both of which are hereby incorporated by reference. In some embodiments, the magnetic resonance imaging moiety and the optical imaging moiety can each be attached to the same or different atoms of a polymeric backbone (e.g., by one or more covalent bonds and/or one or more covalent associations). In these embodiments, the magnetic resonance imaging moiety and the optical imaging moiety can be further linked to one another (i.e., forming a cyclic structure that includes the magnetic resonance imaging moiety, the optical imaging moiety, and the polymeric backbone). In other embodiments, the magnetic resonance imaging moiety and the optical imaging moiety can be linked to one another as described elsewhere and then attached to the polymeric backbone through either the magnetic resonance imaging moiety or the optical imaging moiety. In some embodiments, the probes can further include one or more targeting moieties. For example, the targeting moiety can be selected on the basis of its ability to maximize the likelihood of probe uptake into host response cells (e.g., activated microglia) in the pathology or at its periphery) and/or into the cells of the pathology itself. In certain embodiments, the targeting moiety can be a GABA receptor. Various fluorochromes, polymeric backbones, protective side chains, and targeting moieties are also described in, for example, Weissleder et al., U.S. Patent No. 6,083,486, which is hereby incorporated by reference. In some embodiments, the probes can have a relatively long half-life in the blood pool, e.g., having a half-life in the blood pool of at least about 2 hours (e.g., at least about 6 hours, at least about 12 hours, at least about 20 hours, at least about 30 hours, at least about 40 hours, or at least about one week). In certain embodiments, probes having one or more iron oxide nanoparticles can be useful for this purpose. Magnetic nanoparticles (e.g., having an average particle size of from about 5 nm to about 100 nm) generally have relatively long blood half-lives, because they are generally too large to undergo renal elimination and generally too small to be recognized by phagocytes. Such nanoparticles are eventually internalized, predominantly by cells of the reticuloendothelial system, and the superparamagnetic iron dissolves and joins normal iron pools. An exemplary probe of this type is Cy5.5- CLIO. Alternatively, probes having one or more chelator moieties and a chelated paramagnetic or superparamagnetic metal atoms or ions connected to a polymeric backbone-protective chain combination can also have a relatively long half lives in the blood pool. Exemplary probes of this type have Gd chelates and one or more fluorescent optical imaging moieties attached to, for example, poly(lysine) (e.g., poly(D-lysine), dextran, or PL-MPEG.

Bifunctional Probe Synthesis and Administration In general, the bifunctional probes can be prepared by coupling, for example, a precursor optical imaging moiety to a precursor magnetic resonance imaging moiety. In some embodiments, a precursor optical imaging moiety and a precursor magnetic resonance imaging moiety can each be coupled to a polymeric backbone. Bifunctional probes, precursor optical imaging moieties, precursor magnetic resonance imaging moieties, and polymeric backbones can be obtained commercially or synthesized according to methods described herein and/or by conventional, organic chemical synthesis methods. The probes and probe intermediates described herein can be separated from a reaction mixture and further purified by a method such as column chromatography, high-pressure liquid chromatography, or recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the probes and probe intermediates described herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired probes and probe intermediates. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the probes and probe intermediates described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. In some embodiments, precursor magnetic resonance imaging moieties can include coated magnetic nanoparticles (e.g., cross-linked dextran-coated nanoparticles). The coatings can be further derivatized with one or more nucleophilic (e.g., amino groups) or electrophilic (e.g., activated ester) functional groups. Carboxy functionalized nanoparticles can be made, for example, according to the method of Gorman (see WO 00/61191). In this method, reduced carboxymethyl (CM) dextran is synthesized from commercial dextran. The CM-dextran and iron salts are mixed together and are then neutralized with ammonium hydroxide. The resulting carboxy functionalized nanoparticles can be used for coupling amino functionalized groups. Carboxy-functionalized nanoparticles can also be made from polysaccharide coated nanoparticles by reaction with bromo or chloroacetic acid in strong base to attach carboxyl groups. In addition, carboxy-functionalized particles can be made from amino-functionalized nanoparticles by converting amino to carboxy groups by the use of reagents such as succinic anhydride or maleic anhydride. Nanoparticle size can be controlled by adjusting reaction conditions, for example, by using low temperature during the neutralization of iron salts with a base as described in U.S. Patent No. 5,262,176. Uniform particle size materials can also be made by fractionating the particles using centrifugation, ultrafiltration, or gel filtration, as described, for example in U.S. Patent No. 5,492,814. Nanoparticles can also be synthesized according to the method of Molday (Molday, R.S. and D. MacKenzie, "Immunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells, " J. Immunol. Methods, 1982, 52(3):353-67, and treated with periodate to form aldehyde groups. The aldehyde-containing nanoparticles can then be reacted with a diamine (e.g., ethylene diamine or hexanediamine), which will form a Schiff base, followed by reduction with sodium borohydride or sodium cyanoborohydride. Dextran-coated nanoparticles can be prepared and cross-linked with epichlorohydrin. The addition of ammonia will react with epoxy groups to generate amine groups, see Hogemann, D., et al., Improvement of MRI probes to allow efficient detection of gene expression Bioconjug. Chem. 2000. 11(6): 941-6, and Josephson et al., "High-efficiency intracellular magnetic labeling with novel superparamagnetic- Tatpeptide conjugates, " Bioconjug. Chem., 1999, 10(2):186-91. This material is known as cross-linked iron oxide or "CLIO" and when functionalized with amine is referred to as amine-CLIO or NH₂-CLIO. Carboxy-functionalized nanoparticles can be converted to amino- functionalized magnetic particles by the use of water-soluble carbodiimides and diamines such as ethylene diamine or hexane diamine. The synthesis of NH₂-CLIO and coupling reactions thereof, e.g., with fluorochromes are also described in, for example, Josephson, et al. Bioconjugate Chem. 2002, 13, 554; Kircher, et al, Cancer Res. 2003, 63, 8122; and Kircher, et al, Bioconjugate Chem. 2004, 15, 242, each of which is incorporated herein by reference. The synthesis of probes having metal chelates and fluorescent optical imaging moieties attached to polymeric backbones are described in Huber, M.M. Bioconjug. Chem. 1998, 9, 242-249. The probes of this invention include the probes themselves, as well as their salts and their prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a probe described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate or sulfate) on a probe described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active probe. Pharmaceutically acceptable salts of the probes include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the probes and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄ salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Salt forms of the probes of any of the formulae herein can be amino acid salts of carboxy groups (e.g. L-arginine, -lysine, -histidine salts). The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a subject (e.g., a patient), together with one of the probes described herein, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the probe. Pharmaceutically acceptable carriers, adjuvants, and vehicles that may be used in the new methods include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β- cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of probes described herein. The probes and compositions described herein can, for example, be administered orally, parenterally (e.g., subcutaneously, intracutaneously, intravenously, intramuscularly, intraarticularly, intraarterially, intrasynovially, intrasternally, intrathecally, intralesionally and by intracranial injection or infusion techniques), by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, by injection, subdermally, intraperitoneally, transmucosally, or in an ophthalmic preparation, with a dosage ranging from about 0.01 mg/Kg to about 1000 mg/Kg, (e.g., from about 0.01 to about 100 mg/kg, from about 0.1 to about 100 mg/Kg, from about 1 to about 100 mg/Kg, from about 1 to about 10 mg/kg). The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep. 50, 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970). The compositions described herein may include any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles in addition to any of the probes described herein. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non- toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. The compositions described herein may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols. Topical administration of the compositions is useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the compositions can be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The compositions can also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches are also included in this invention. The compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. A composition optionally having the probe and an additional agent (e.g., a therapeutic agent or delivery or targeting agent) can be administered using an implantable device. Implantable devices and related technologies are known in the art and are useful as delivery systems where a continuous, or timed-release delivery of compounds or compositions delineated herein is desired. Additionally, implantable device delivery systems can be useful for targeting specific points of compound or composition delivery (e.g., localized sites, organs). See, e.g., Negrin et al., Biomaterials, 22(6):563 (2001). Timed-release technology involving alternate delivery methods can also be used in the new methods. For example, timed-release formulations based on polymer technologies, sustained-release techniques and encapsulation techniques (e.g., polymeric, liposomal) can also be used for delivery of the compounds and compositions delineated herein. EXAMPLES The invention is further illustrated by the following Examples. The Examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the invention in any way. To demonstrate the ability of bifunctional probes to act as a contrast agent for pre-operative brain tumor imaging, MRI was performed on brain tumor bearing rats using Cy5.5-CLIO as the candidate probe.

Example 1. Synthesis of Cy5.5-CLIO The Amino-CLIO nanoparticle (see e.g., Josephson, L., High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjug Chem, 10: 186-191., 1999; and Josephson, L., Perez, J. M., and Weissleder, R. Magnetic nanosensors for the detection of oligonucleotide sequences. Angewandte Chemie, International Edition, 40: 3204-3206, 2001) at 0.2 mL at 10 mg Fe/mL in 0.02M Citrate pH 8, was added to a tube of Cy5.5 (Amersham-Pharmacia) at room temperature for 2 hours, with subsequent incubation at 4°C overnight. Unreacted Cy5.5 was removed by gel filtration (Sephadex G-25 in 0.02 M Citrate, 0.15 M NaCl, pH 8). The number of dyes per crystal was obtained from the absorbance at 675 nm using an extinction coefficient of 250,000 M 'cm^"1 for Cy5.5. Iron concentration was assayed as described in Josephson, L., et al., High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjug Chem, 10: 186-191, 1999; and the concentration of nanoparticles obtained by assuming 2064 iron atoms per crystal (see, e.g., Shen, T., et al., Monocrystalline iron oxide nanocompounds (MION): physicochemical properties. Magn Reson Med, 29: 599-604, 1993). The nanoparticle, Cy5.5-CLIO, was 32 nm using the volume estimation of laser light scattering (Malvern Instruments) and had an average of 1 Cy5.5 dye per crystal. Example 2. Animal Model A 9L rat gliosarcoma cell line stably transfected to express green fluorescence protein (GFP) (see, e.g., Moore, A., et al., Novel gliosarcoma cell line expressing green fluorescent protein: A model for quantitative assessment of angiogenesis. Microvasc Res, 56: 145-153, 1998) was cultured at 37°C in a humidified 5% CO₂ atmosphere in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 500 μg/ml Geniticin G418 (all products from Cellgro, Herndon, VA). The medium was changed every 3 days, and cells were passaged once per week (1 :10 split ratio). Eight Fisher 344 rats (Charles River Laboratories, Wilmington, MA), 200-250 grams, were anesthetized with ketamme/xylazine (i.p., 65/10 mg/kg) and immobilized in a stereotactic frame. A linear skin incision was made over the bregma, and a 1 mm diameter burrhole was drilled into the skull 3 mm posterior and 3 mm lateral to the bregma. A 10-μl gas-tight syringe (Hamilton, Reno, NV) was then used to inject 5 μl of the 9L-GFP-cell suspension (10⁶ cells in HBSS) in the striatum at a depth of 3 mm from the dural surface. The injection was done slowly over 5 minutes, and the needle was withdrawn over another 10 minutes. The burrhole was occluded with bone wax (Ethicon, Sommerville, NJ) to prevent leakage of cerebrospinal fluid, and the skin was closed with nonmagnetic sutures. MR imaging of rats was performed 10-14 days after tumor inoculation, when the tumors had reached diameters of approximately 2-5 mm, 24 hours after i.v. injection of 15 mg Fe/kg Cy5.5-CLIO.

Example 3. MR Imaging Animals were injected with 15 mg/kg body weight Cy5.5-CLIO via tailvein injection. After 24 hours, MR imaging was performed at 4.7T (Bruker Instruments, Billerica, MA) equipped with a 30 gauss/cm gradient set and a 37 mm diameter birdcage coil resonating at 200 MHz. Multiple slice multiple echo Proton-density/T2- weighted (TR/TE 15, 30, 45, 60/2000) spin echo sequences were obtained using 2 NEX, a 256 x 256 matrix, a 3.0 cm field of view (resulting in an in-plane resolution of 117 μm), a slice thickness of 1 mm and a total imaging time of 17 min. For MRI- histology correlation, rats were sacrificed (pentobarbital, i.p. 200 mg/kg), perfused with 250 ml phosphate-buffered saline and brains were stained (hematoxylin-eosin and DAB amplified Prussian Blue).

Example 4. Optical Imaging Twenty- four hours after i.v. injection of 15 mg/kg body weight Cy5.5-CLIO a craniotomy was performed to operatively expose the tumor and surrounding tissue. Noninvasive optical imaging was performed using a custom built surface reflectance imaging system (Siemens Medical Systems, Erlangen, Germany), based on a multichannel imaging system design (see, e.g., Mahmood, U., et al. Feasibility of in vivo multichannel optical imaging of gene expression: experimental study in mice. Radiology, 224: 446-451, 2002). The system is capable of near simultaneous data acquisition in four channels, including a broad spectrum visible white light similar as seen by the un-aided eye, an excitation/emission filter set for GFP imaging, and a filter set for Cy5.5 imaging. Images were acquired with an exposure time of 400 msec, 500 msec, and 200 msec for the GFP, Cy5.5 and white light channels, respectively.

Example 5. Histology The distribution of tumor in the brain was determined by GFP fluorescence and Cy5.5 fluorescence 24 hours after i.v. injection of 15 mg/kg body weight Cy5.5- CLIO. Tumors were cryo-sectioned and fluorescence microscopy of GFP and Cy5.5 fluorescence was performed on air-dried sections using an inverted epifluorescence microscope (Axiovert 100; Zeiss, Thornwood, NY). A cooled charge-coupled device camera (Sensys; Photometries, Tucson, AZ) was used for image capture. Sections were subsequently stained with hematoxylin-eosin and DAB amplified Prussian Blue and examined with bright light microscopy. Slices were also examined by laser-scanning confocal microscopy using a Zeiss LSM 5 Pascal. Glial cells were identified immunohistochemically using a primary monoclonal mouse-anti-rat Ab against CD1 lb (Serotec, Raleigh, NC) and a secondary rhodamine labeled rabbit anti-mouse Ab (Jackson Immnunoresearch Laboratories, West Grove, PA). The fluorescence from GFP (tumor), Cy5.5 (nanoparticle) and the rhodamine (glia cells and macrophages) were obtained by selecting appropriate excitation and emission settings.

Example 6. Accuracy of Determining Tumor Extent via Cy5.5 Fluorescence Five brain tumors were cryosectioned into 4-8 slices each, resulting in 25 slices (20 μm slice thickness with an interleave of 500 μm), and digital images of tumors and surrounding brain tissue captured using an inverted fluorescence microscope equipped with a CCD camera (Zeiss Axiovert 100). Regions of interest were placed on digitized images around borders of the tumor as defined by GFP and Cy5.5 fluorescence using CMIR-Image (developed in Interactive Data Language (Research Systems Inc, Boulder, Colorado). The area of each region of interest on each slice was computed and the values of Cy5.5 and GFP positive areas plotted with a linear regression analysis using Microsoft Excel. Example 7. Preoperative MR Imaging Using Cy5.5-CLIO Proton density-weighted and T2 weighted images of a tumor bearing rat brain are shown in FIGS. 1 A and IB, respectively, after the administration of Cy5.5-CLIO. The hypointense tumor relative to the surrounding tissue on T2 -weighted images (FIG. IB) is indicative of nanoparticle accumulation, which causes reduction in signal intensity with T2 weighted spin echo pulse sequences. Reduction in signal intensity on T2 weighted, but not proton density weighted, images is characteristic of monodisperse superparamagnetic iron oxide nanoparticles and is not seen with larger magnetic particles or gadolinium chelates (see, e.g., Rogers, J., Lewis, J., and Josephson, L. Use of AMI-227 as an oral MR contrast agent. Magn Reson Med, 12: 631-639, 1994). The tumor bearing rat brains were subsequently cryo-sectioned coronally, with a slice orientation corresponding to that of the MR image, and histology was performed. There was a relatively high congruency between the signal reduction obtained on MR images and the tumor margins as determined by Hematoxylin-Eosin staining (see FIG. 1C). DAB amplified Prussian blue stain (see FIG. ID) demonstrated the accumulation of iron in the tumor. Staining was negative animals not administered with Cy5.5-CLIO (data not shown).

Example 8. Intra-Operative MR Imaging Using Cy5.5-CLIO A craniotomy was performed on a Fischer 344 rat bearing a 3-mm diameter 9L glioma tumor. FIG. 2 A is an image of a rat after craniotomy and exposition of the tumor in the white light channel after the administration of Cy5.5-CLIO. FIG. 2B is an image of the rat after craniotomy and exposition of the tumor in the green fluorescence protein (GFP) channel after the administration of Cy5.5-CLIO. The GFP channel serves as the gold standard for delineation of true tumor extent. FIG. 2C is an image of the rat after craniotomy and exposition of the tumor in the in the Cy5.5 channel after the administration of Cy5.5-CLIO. Even with exposure times as short as 500 milliseconds, Cy5.5 fluorescence was obtained in an amount to clearly visualize the tumor, as indicated by the correlation with the tumor margin extent as determined by the gold standard GFP fluorescence (see FIG. 2B). The relationship between tumor and Cy5.5 fluorescence was further examined by histology, using epifluorescence microscopy as shown in FIGS. 2D, 2E, and 2F. Hematoxylin-eosin staining (FIG. 2D) was compared with GFP fluorescence (FIG. 2E) and Cy5.5 fluorescence (FIG. 2F). To compare the accuracy of tumor margin delineation by Cy5.5-CLIO uptake with tumor margin delineation by GFP fluorescence, region of interest (ROI) analysis of Cy5.5 and GFP positive areas on 25 slices from five brain tumors was performed (see Examples). The areas obtained from these ROIs were fitted with a linear regression analysis for all 25 slices as shown in FIG. 3. The data showed an excellent fit to a linear equation with a slope of 1.013, an intercept of 0.820 mm² and a R² of 0.996. The slightly higher estimate of tumor volumes obtained with Cy5.5 is due to the uptake of Cy5.5-CLIO by microglia, as described elsewhere. Triple-channel laser scanning confocal microscopy was performed to determine the cellular distribution of Cy5.5-CLIO. FIGS. 4A, 4B, and 4C compare the distribution of Cy5.5-CLIO (Cy5.5 channel, (FIG. 4A)) with the distribution of tumor cells (GFP channel (FIG. 4B)) and microglia and macrophages (CD1 lb immunohistochemistry using the rhodamine channel (FIG. 4C)). Cy5.5-CLIO was strongly associated with CD1 lb positive cells (FIG. 4D) and much less with GFP positive cells (FIG. 4E). The tumor border as defined by tumor cells was then determined in the GFP channel (see FIG. 4F) and by microglia (CD1 lb staining, FIG. 4G) with confocal microscopy. As the overlay FIG. 4H shows, the presence of microglia exhibits relatively high congruency with GFP fluorescence at the tumor-brain interface.

Microglia consistently extended slightly beyond the tumor border as defined by GFP fluorescence (FIG. 4H), consistent with the higher estimation of tumor area by Cy5.5 fluorescence as described in FIG. 3. Example 9. Cellular uptake of Cy5.5-CLIO in Cell Lines Cellular uptake of Cy5.5-CLIO has also been demonstrated in culture for the following cell lines: U937 cells (macrophage line, see FIG. 5), C6 cells (brain tumor, see FIG. 6), Gli36 cell Iine-Cy5.5 (brain tumor, see FIG. 7), LLC cell Iine-Cy5.5 (Lewis lung carcinoma, see FIG. 8), and HT29 cell line-CLIO-Cy5.5 (colon cancer cells, see FIG. 9). FIGS. 5-9 show relative number of cells vs. fluorescence signal intensity revealing cellular uptake of probe in culture. "Control" cells were not exposed to probe. Cells exposed to probe at a concentration of 100 mgFE/ml demonstrate uptake reported as increased fluorescence compared to control cells. The macrophage line (a host response cell line) demonstrates a greater uptake than the other lines tested (see FIG. 5). The data shown in FIGS. 5-9 was obtained according to protocols described in Neoplasia, 2002 May-Jun, 4(3):228-36. Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment. Bogdanov AA Jr, Lin CP, Simonova M, Matuszewski L, Weissleder R. Example 10. Tumor Margin Delineation FIGS. 10A-10C (Gli36 brain tumor), 10E-10G (9L brain tumor), and 10J-10L (C6 brain tumor) corresponds to images of each tumor type under the white light, GFP, and Cy5.5 channels, respectively. Normalized histograms (FIGS. 10D, 10H, and 10M) of surface signal of GFP and Cy5.5 fluorescence demonstrates that improved tissue penetration at wavelengths in the far red and NIR compared to GFP allow the determination of tumor location even at a distance from the surface. The data shown in FIGS. 10A-10M was obtained according to protocols described in Cancer Res., 2000, Sep l ;60(17):4953-8. In vivo imaging of proteolytic enzyme activity using a novel molecular reporter.Tung CH, Mahmood U, Bredow S, Weissleder R. FIGS. 11 A-l 1C show high magnification of tumor border demonstrates relative area on a histological slide determined by Cy5.5 fluorescence representing area of probe uptake vs. area determined by GFP (representing "true" tumor area, since only the tumor cells used in this experiment express GFP). The high correlation suggests that the probe is distributed to a volume that highly approximates and minimally overestimates true tumor margins. The data shown in FIGS. 11 A-l 1C was generated using similar protocols to those used to generate the data presented in FIG. 3.

OTHER EMBODIMENTS A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS :

1. A method for differentiating a diseased tissue from normal host tissue in a subject, the method comprising: (a) administering a bifunctional probe to the subject, wherein the probe comprises an optical imaging moiety and a magnetic resonance imaging moiety; (b) obtaining a preoperative magnetic resonance image of the diseased tissue and the host tissue, wherein the portion of the magnetic resonance image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins of the diseased tissue. (c) removing or displacing one or more layers of cutaneous matter, subcutaneous matter, skeletal matter, vascular tissue or host tissue, thereby partially or fully exposing the diseased tissue and host tissue; and (d) obtaining at least one optical image of the exposed diseased tissue and host tissue during and/or after step (c), wherein for each optical image obtained, the portion of the image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins or changes in the margins of the diseased tissue in response to step (c).

2. A method for differentiating a diseased tissue from normal host tissue in a subject under intra-operative conditions, the method comprising: (a) administering to the subject a probe comprising an optical imaging moiety, wherein the probe is preferentially sequestered into one or more host response cells in the diseased tissue or at its periphery; (b) removing or displacing one or more layers of cutaneous matter, subcutaneous matter, skeletal matter, vascular tissue or host tissue, thereby partially or fully exposing the diseased tissue and host tissue; and (c) obtaining at least one optical image of the exposed diseased tissue and host tissue during and/or after step (b), wherein for each optical image obtained, the portion of the image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins or changes in the margins of the diseased tissue in response to step (b).

3. The method of claim 1 or 2, wherein the method further comprises the steps of: (d) resecting the diseased tissue; and (e) obtaining at least one optical image of the exposed diseased tissue and host tissue during step (d), wherein for each optical image obtained, the portion of the image associated with the diseased tissue: (i) visually contrasts with the portion of the image associated with the host tissue; and (ii) delineates the margins or changes in the margins of the diseased tissue in response to step (d).

4. The method of claim 1 or 2, wherein the host tissue is brain tissue.

5. The method of claim 1 or 2, wherein the host tissue is breast tissue.

6. The method of claim 1 or 2, wherein the host tissue is a sentential lymph node.

7. The method of claim 1 or 2, wherein the host tissue is gastrointestinal tissue.

8. The method of claim 1 or 2, wherein the diseased tissue is a tumor.

9. The method of claim 1 or 2, wherein the probe has a half-life in the blood pool of at least about 2 hours.

10. The method of claim 9, wherein the probe has a half-life in the blood pool of at least about 6 hours.

11. The method of claim 9, wherein the probe has a half-life in the blood pool of at least about 12 hours.

12. The method of claim 9, wherein the probe has a half-life in the blood pool of at least about 20 hours.

13. The method of claim 9, wherein the probe has a half-life in the blood pool of at least about 30 hours.

14. The method of claim 9, wherein the probe has a half- life in the blood pool of at least about 40 hours.

15. The method of claim 1 , wherein the magnetic resonance imaging moiety comprises a magnetic nanoparticle.

16. The method of claim 15, wherein the magnetic nanoparticle comprises a magnetic metal oxide.

17. The method of claim 16, wherein the magnetic metal oxide is a superparamagnetic metal oxide.

18. The method of claim 17, wherein the metal oxide is iron oxide.

19. The method of claim 15, wherein the nanoparticle is an amino- derivatized cross-linked iron oxide nanoparticle.

20. The method of claim 15, wherein the nanoparticle has an average diameter of from about 5 nm to about 100 nm.

21. The method of claim 20, wherein the nanoparticle has an average diameter of from about 20 nm to about 80 nm.

22. The method of claim 20, wherein the nanoparticle has an average diameter of from about 40 nm to about 60 nm.

23. The method of claim 1 or 2, wherein the optical imaging moiety comprises at least one fluorochrome.

24. The method of claim 23, wherein the fluorochrome is a near infrared fluorochrome.

25. The method of claim 24, wherein the near infrared fluorochrome comprises Cy5.5, Cy5, Cy 7, Alexa Fluoro 680, Alexa Fluoro 750, IRD41, IRD700, NIR-1, LaJolla Blue, indocyanine green (ICG), indotricarbocyanine (ITC), or a chelated lanthanide compound.

26. The method of claim 24, wherein the fluorochrome is Cy5.5.

27. The method of claim 1, wherein the optical imaging moiety and the magnetic resonance imaging moiety are covalently linked.

28. The method of claim 27, wherein the optical imaging moiety and the magnetic resonance imaging moiety are covalently linked by a bond between a carbonyl group on the optical imaging moiety and an amino group on the magnetic resonance imaging moiety.

29. The method of claim 1, wherein the probe further comprises a polymeric backbone, wherein each of the optical imaging moiety and the magnetic resonance imaging moiety are linked to the polymeric backbone.

30. The method of claim 29, wherein each of the optical imaging moiety and the magnetic resonance imaging moiety are covalently linked to the backbone.

31. The method of claim 29, wherein the magnetic resonance imaging moiety comprises a chelator moiety and a chelated paramagnetic or superparamagnetic metal atom or ion.

32. The method of claim 31 , wherein the ion is Gd .

33. The method of claim 31 , wherein the chelator is selected from the group consisting of 1,4,7, 10-tetraazacyclodo-decane-N,N',N",N'"-tetraacetic acid; o 1 ,4,7, 10-tetraaza-cyclododecane-N,N',N"-triacetic acid; 1 ,4,7-tris(carboxymethyl)- 10- (2'-hydroxypropyl)-l,4,7,10-tetraazocyclodecane; l,4,7-triazacyclonane-N,N',N"- triacetic acid; 1,4,8,1 l-tetraazacyclotetra-decane-N,N',N",N'"-tetraacetic acid; diethylenetriamine-pentaacetic acid (DTP A); ethylenedicysteine; bis(aminoethanethiol)carboxylic acid; triethylenetetraamine-hexaacetic acid;5 ethylenediamine-tetraacetic acid (EDTA); l,2-diaminocyclohexane-N,N,N',N'- tetraacetic acid; N-(hydroxy-ethyl)ethylenediaminetriacetic acid; nitrilotriacetic acid; and ethylene-bis(oxyethylene-nitrilo)tetraacetic acid.

34. The method of claim 29, wherein the polymeric backbone comprises a0 polysaccharide, a nucleic acid, polypeptide or a synthetic polymer.

35. The method of claim 34, wherein the polymeric backbone further includes at least one protective chain covalently linked to the backbone. 5

36. The method of claim 35, wherein the protective chain comprises polyethylene glycol, methoxypolyethylene glycol, methoxypolypropylene glycol, copolymers of polyethylene glycol and methoxypolypropylene glycol, dextran, or polylactic-polyglycolic acid. 0

37. The method of claim 1 or 2, wherein the diseased tissue is associated with or causes changes in one or more microglial cells.

38. The method of claim 37, wherein substantially all of the probe is sequestered by the microglial cells.5

39. The method of claim 1 or 2, wherein the portion of the image associated with the host tissue represents a tissue that is substantially free of the diseased tissue.

40. The method of claim 1 or 2, wherein the probe further comprises a GABA receptor.

41. The method of claim 1 , wherein the diseased tissue margins delineated in the magnetic resonance image are substantially congruent with the diseased tissue margins delineated in at least one of the optical images obtained in step (c).

42. The method of claim 1 or 2, wherein the subject is a mammal.

43. The method of claim 42, wherein the mammal is a human.

44. The method of claim 2, wherein the probe further comprises a nanoparticle.

45. The method of claim 44, wherein the nanoparticle is an iron oxide nanoparticle.

46. A computer readable medium on which is recorded one or more magnetic resonance images and/or one or more optical images that differentiate a diseased tissue from normal host tissue in a subject, wherein the optical image is generated by a process comprising the method of claim 1 or claim 43.

47. Use of a bifunctional probe comprising an optical imaging moiety and a magnetic resonance imaging moiety to differentiate a diseased tissue from normal host tissue in a subject under intra-operative conditions to remove or explore the diseased tissue.

48. Use of a bifunctional probe comprising an optical imaging moiety and a magnetic resonance imaging moiety in the manufacture of a medicament to differentiate a diseased tissue from normal host tissue in a subject under intra- operative conditions to remove or explore the diseased tissue.