Radiographic imaging modalities such as CT scan and MRI, although useful for delineating the deep extent of advanced carcinomas (4), are not sufficiently sensitive to detect small, earlier intraepithelial lesions which are more readily cured. Optical imaging is a relatively new modality which enables real time, non-invasive, high resolution imaging of epithelial tissue (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Optical imaging of tissue can be carried out noninvasively in real time, yielding high spatial resolution (less than 1 micron lateral resolution). Optical imaging systems are inexpensive, robust and portable because of advances in computing, fiber optics and semiconductor technology. Confocal microendoscopes which image near infra-red (NIR) reflected light have been used to image sub-cellular features in epithelial tissue at video rate to depths exceeding 400 microns. Optical imaging systems are ideally suited for early detection of intraepithelial disease and to assess tumor margins and response to therapy.

This paper describes a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. Our group is working to develop optically active contrast agents that can be applied topically to areas of tissue at risk to monitor the three-dimensional profile of the targeted biomarkers as well as morphologic and architectural biomarkers such as nuclear to cytoplasmic ratio. We believe these contrast agents and imaging systems will have broad applicability to detect and monitor many types of cancer.

Here we describe the general approach our group has taken to push forward toward molecular characterization of epithelial cancers and present initial results obtained using optically active contrast agents to image the expression of three well characterized molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. This same approach can be used to develop contrast agents to image the expression of promising new biomarkers. For example, Serial Analysis of Gene Expression (SAGE) libraries can be used to identify novel targets for contrast agent development from the pool of genes differentially expressed in early neoplasia (22, 23, 24). Alternatively, in vivo phage display can be used to identify peptides that specifically bind to the surface of neoplastic cells and tumor vascular endothelium in target organ sites (25, 26). Discovering new biomarkers and developing techniques to image their expression in vivo could be particularly useful for monitoring response to therapy.

At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. We are developing systems to image both reflected light and fluorescent light at two spatial scales: (i) confocal microscopy, with micron resolution to image cell morphology from a small field of view and (ii) multi-spectral digital imaging with mm resolution to image tissue morphology from large fields of view. These systems can assess both native optical contrast as well as that afforded by optically active contrast agents. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo. Figure 1 illustrates our overall approach.

Figure 1: Approach to the optical molecular imaging of cancer.

Initial Biomarker Selection

A number of cancer biomarkers have already been identified, and these form the basis of our initial work. Cancer results from accumulation of a series of key mutations in expanding clones of cells. Genetic mutations lead to oncogene activation, loss of tumor suppressor gene function and/or abnormal DNA repair mechanisms (Table I). These mutations lead to changes in the gene expression profile and cellular phenotype which are manifested in alterations in cell metabolism and morphology, protein secretion, cytoskeletal properties, etc. at the cellular level and in cell-cell and cell-stromal interactions in tissue. Work by Condeelis, Jain, and Coffee provides important connections between specific genes and proteins and related changes in chromatin density (27), tumor metastases (28, 29, 30), host cell-tumor cell interactions (31), and angiogenesis (32, 33). It has been shown that these molecular events play a major role in cancer progression and can be valuable biomarkers for cancer diagnosis, grading and prognosis (34, 35). A better understanding of the molecular mechanisms of carcinogenesis has inspired novel molecular oriented strategies for cancer therapy (Table I). Currently these molecular events cannot be imaged in patients – the goal of the work described in this paper is to develop the contrast agents and imaging systems that can be used in vivo to address this important need.


It has been suggested that there are six acquired capabilities shared by most human cancers which collectively dictate malignant growth: (i) self-sufficiency with respect to growth signals, (ii) insensitivity to growth-inhibitory signals, (iii) evasion of programmed cell death, (iv) limitless replicative potential, (v) sustained angiogenesis, and (vi) tissue invasion and metastasis (36). The first three capabilities can be combined as a class of factors that lead to an uncoupling of a cell’s growth program from environmental signals (36). In our work, we have initially targeted biomarkers related to these capabilities: EGFR: uncoupling of a cell’s growth program from environmental signals; HPV related oncoproteins: driving increased proliferation; and MMPs: enabling tissue invasion and metastasis.

EGFR: ERB B1 initially was discovered as one of two oncogenes carried by the avian erythroblastosis virus; the corresponding proto-oncogene was found to encode a membrane-associated tyrosine kinase protein that was identified later as the EGF receptor (37, 38, 39). There is good consensus among immunohistochemical studies in the cervix that EGFR levels demonstrate a statistically significant increase as lesion severity progresses from earlier dysplasia to invasion (40, 41, 42, 43). Similarly, EGFR expression was shown to increase in oral cavity epithelium as the tissue undergoes a multi-step tumorigenesis (44, 45). EGFR is over-expressed in at least 45% of non-small cell lung cancers (NSCLC), and remains an intensively studied target for drug inhibition due to its correlation with a poor prognosis when accompanied by other cancer biomarkers (46).

MMPs: At late stages of tumor progression tumor cells invade adjacent tissue and travel to distant sites to form metastases. Metastases cause 90% of human cancer death (47). MMPs enable invasion and metastasis and are implicated in other activities important for tumor growth, such as angiogenesis and growth signaling (36). It has been demonstrated in many studies that MMPs (MMP 1, 2, 3, 9, 10, 11, 13, MT1-MMP) are all expressed in oral cancer and have roles in tumor progression [(48) and references therein]. In cervix, five studies showed progressive increases in staining for MMPs as tissue progressed from normal to SIL to cervical carcinoma (49, 50, 51, 52, 53). MMP staining progressively increased as the grade of the lesion or stage of the tumor increased. Sienel showed homogeneous expression of MMP-9 and MMP-2 can serve as significant indicators of poor patient survival in lung carcinomas, as well (54, 55). MMP-9 was overexpressed by a factor of 2, while active MMP-2 was overexpressed 17 times in a population of 36 lung cancer patients (56).

HPV Related Oncoproteins: Since the mid-1970s, substantial evidence has accumulated supporting the role of some types of HPV in the etiology of at least 95% of cases of cervical neoplasia (57). There are more than 100 HPV genotypes; about 20 of these have oncogenic potential (58). Approximately 75% of HGSILs are infected with either HPV 16, 18 or a member of the 30s group (58). In high-grade lesions and cancers, viral genomes are frequently integrated into chromosomes of the host cells (59). The unregulated production of viral replication proteins (especially products of the viral E6 and E7 genes) tends to drive the host cell into S phase, thereby helping to generate a cancer. The E6 protein appears to alter cell growth through effects on p53, an endogenous tumor-suppressor protein (58, 60). The HPV 16 or 18 E6 proteins target p53 for degradation, resulting in a loss of p53 activity within cells (58). In normal cells, following DNA damage p53 induces G1 phase growth arrest to allow the cell to repair the damage before progressing to the S phase and undergoing DNA synthesis (60). However, in cells infected with high-risk HPV types, reduced p53 levels may lead to unregulated cell cycle progression and allow accumulation of genetic mutations. The E7 protein of HPV 16 binds to retinoblastoma protein (pRb) and its related proteins. When E7 binds to pRb, it prevents it from binding to its normal associates in the cell and leads to increased transcription of molecules that drive cell proliferation.

Types of Contrast Agents

Our approach to contrast agent development is illustrated in Figure 2. The agents consist of three parts: (i) a probe molecule which provides molecular specific recognition of cancer biomarkers conjugated to (ii) an optically interrogatable label in (iii) a mucoadhesive, permeation enhancing formulation. In our work, we are testing three different types of optically active labels, including metal nanoparticles, quantum dots and organic fluorescent dyes. We are pursuing two types of molecular probes: monoclonal antibodies against cancer specific biomarkers and peptides which bind selectively to cancer specific biomarkers. With this approach, we can significantly expand the number of molecular changes that can be probed using optical imaging. In this paper, we describe contrast agents based on metal nanoparticles, organic fluorescent dyes and quantum dots coupled to monoclonal antibodies against cancer related biomarkers.

Optical Imaging System Development

We are developing two types of novel optical imaging systems to rapidly and non-invasively image the distribution of these agents in vivo. Confocal microscopy is a tool to image tissue noninvasively with subcellular spatial resolution. At the same time, we are developing systems to image a large field of view with limited spatial resolution. We envision that integrating these two types of imaging systems will allow us to examine field effects in the epithelium at risk in these three organ sites, indicating the areas within this field which should be imaged at high resolution with confocal microscopy.

Miniature Confocal Microscopes

In vivo confocal microscopy is a new technology that can provide detailed images of tissue architecture and cellular morphology in living tissue in near real time. The optical sectioning principle of confocal imaging is illustrated in Figure 3. The illumination light (solid line) is focused to a point in the sample. Light produced at the focal region (solid line) is refocused by the lens and partially reflected by the beam splitter to a point at the conjugate image plane. If a small aperture is centered on the focused beam in the conjugate image plane, a majority of light returning from the focal region is passed to the detector. Light generated from outside the focus region (dashed line) is spread out when it reaches the pinhole, so the pinhole aperture significantly reduces its intensity and thereby increases resolution. Scanning the focal spot in the axial and radial dimensions forms an image of reflectance from the focal region of each point in the sample. In epithelial tissue, 1 micron resolution has been achieved with a 200-400 micron field of view and penetration depth up to 500 microns (5, 6, 7, 8, 9, 10, 11, 19, 20, 61, 62).


Figure 3: Schematic of confocal technique.
Our group developed a real-time reflectance based confocal microscope to assess cell morphology and tissue architecture in vivo. A non-fiber optic version of this system showed promise to detect changes associated with cervical precancer (19, 63). Figure 4 shows image pairs of normal (left) and abnormal (right) cervical biopsies from several patients and the corresponding histology. In each pair, the difference in nuclear density and area is evident between the colposcopically normal and abnormal biopsy. Architectural similarities are also evident between confocal and histologic images. The nuclear to cytoplasmic (N/C) ratio extracted from confocal images gave best discrimination between high grade precacnerous lesions and non-high grade lesions. The sensitivity and specificity of this classification are 100% and 91% respectively, much higher than the sensitivity and specificity of clinical impression compared (91% and 62%).

Figure 4: Images from normal (left) and abnormal (right) biopsy pairs from Patient 9 (a-d), Patient 12 (e-h) and Patient 20 (i-l). Increased nuclear density can be seen in the confocal images of the abnormal samples (c, g, k). The confocal images were taken 50 microns below the surface. The histologic images were classified as normal (b, f, j), CIN II/III (d, h) and cancer (l). Scale bars in the confocal images are 50 microns and 100 microns in the histology images.

Fiber optic confocal microscopes are needed to obtain images clinically (64). These instruments are designed to be inserted through a speculum, catheter, large-bore needle, or biopsy channel of an endoscope. We developed a fiber optic reflectance confocal microscope (Fig. 5) (65, 66, 67). This system is currently being tested in vivo to image precancerous lesions in the uterine cervix and the oral cavity at the UT MD Anderson Cancer Center. Results indicate that N/C ratio can be imaged in vivo in real time with this system.



Figure 5: Schematic of the fiber optic confocal microscope.
Alternatively, confocal microscopes can be constructed to image both reflectance and fluorescence. We have constructed a confocal microendoscope that employs a Digital Mirror Device (DMD) to illuminate fiber-core patterns. The novel idea in this system is the application of a DMD (68) illustrated in Figure 6, to improve the contrast, confocal sectioning ability, and optical efficiency (69). A 30,000-fiber image guide was positioned in the object plane of a conventional microscope configured for reflected light illumination. The axial FWHM is 1.6μm. A dysplastic bronchial fragment imaged confocally using 1μm spatial sampling is illustrated in Figure 7. Images were acquired in reflectance and fluorescence modes (without the fiber bundle). The fragment is from freshly excised tissue. No stains or contrast agents were employed. At this resolution it is clearly possible to identify tissue architecture and the spatial arrangement of cells. We hypothesize that it will be possible to realize images of similar quality in a confocal microendoscope if the DMD is employed to enhance the contrast by illuminating and/or detecting light from patterns of fiber cores.



Figure 6: Block diagram of the DMD based confocal microscope.


Figure 7: Part (a) shows the autofluorescence image while part (b) shows a pseudo-color image composed of the autofluorescence signal (green), tissue reflectance at the same level (blue), and tissue reflectance 10μm lower (red).

Multi-spectral Digital Imaging Systems

Confocal microscopes can image cell morphology and tissue architecture in three dimensions in vivo. However, the field of view of these systems is limited, and it would be impractical to scan the surface of even a reasonably small organ like the uterine cervix at 1 micron spatial resolution and a hundreds-of-microns field of view. Instead we envision coupling the microscopes with lower-resolution imaging systems that can provide images of the epithelial surface at risk and can be used to scan even large organ sites. Figure 8 illustrates the concept and recent results to image autofluorescence and reflectance using an inexpensive (<$500), commercially available, video-rate, color CCD camera (70). A colposcope is essentially a low power stereo microscope mounted on a stand. In colposcopy, a health care provider visually examines pattern of white light reflected from the cervix before and after application of acetic acid. We extended the ability of the colposcope to measure tissue autofluorescence, which has the potential to increase both specificity and sensitivity of the procedure and to guide where higher resolution images should be obtained. In our current work, we explored the use of non-specific contrast agents such as acetic acid, in conjunction with the multi-spectral digital colposcope to indicate regions of tissue which should be probed in greater detail (70). We believe this approach can also be used to image tissue following topical application of molecular specific contrast agents.

Optical Contrast Agent Development

Metal Nanoparticles

Gold nanoparticles have been extensively used as molecular specific stains in electron microscopy of cells and tissues (71, 72). As result, the fundamental principle of interactions between gold particles and biomolecules, especially proteins, have been thoroughly studied. However, colloidal gold nanoparticles also exhibit beautiful and intense colors in the visible and NIR spectral regions. These colors are the result of excitation of surface plasmon resonances in the metal particles and are extremely sensitive to the sizes, shapes, and aggregation state of the particles, to the dielectric properties of the surrounding medium and to adsorption of ions on the surface of the particles (73).

The potential of using metal nanoparticles as optically interrogatable biological labels has recently been recognized, leading to development of a variety of novel applications in bioanalytical chemistry with unprecedented sensitivity (74, 75, 76, 77, 78, 79). Among all the interesting properties of metal nanoparticles, the ability to resonantly scatter light at frequencies coinciding with the particles’ surface plasmon resonances is still to be explored for in vivo biological applications. This property can be used to develop contrast agents for in vivo reflectance. The scattering cross section of gold nanoparticles is extremely high compared to polymeric spheres of the same size (Fig. 9), especially in the red. This property is critical for development of contrast agents for optical imaging in living organisms because light penetration depth in tissue dramatically increases with increasing wavelength in the near infrared. Another interesting optical property of gold nanoparticles that can be exploited for vital optical imaging is the increase in scattering cross section per particle when the particles agglutinate (Fig. 9, right). These changes produce a large optical contrast between isolated gold particles and assemblies of gold particles. This increase in contrast improves the ability to image markers which are not uniquely expressed in diseased tissue, but are expressed at higher levels relative to normal tissue (such as EGFR), and to develop highly sensitive labeling procedures which do not require intermediate washing steps to remove single unbound particles.

Figure 9: (Left) The wavelength dependence of visible light scattering by suspensions of polystyrene spheres and gold nanoparticles with the same concentration. (Right) compares scattering of isolated and closely agglutinated conjugates of 12 nm gold nanoparticles with monoclonal antibodies for EGFR.

We are developing contrast agents based on the conjugation of gold nanoparticles with monoclonal antibodies to the epidermal growth factor receptor (EGFR) (80), metallo-proteases 2 and 9 (MMP 2 and MMP 9) and the E7 protein associated with HPV 16. In these studies we used gold nanoparticles with ca. 12 nm in diameter. This size is approximately the same as the size of antibodies which are routinely used for molecular specific labeling and targeting. In our preliminary data, gold conjugates are stable in biological samples and we do not observe non-specific aggregation at any stage of evaluation.

Metal Nanoparticles and EGFR

Figure 10 shows confocal reflectance images and combined transmittance/reflectance images of SiHa cells labeled with anti-EGFR/gold conjugates. Comparison of the labeling pattern with transmittance images of the cells indicates that labeling predominately occurs on the surface of the cytoplasmic membrane. The labeling pattern is consistent with the fact that the monoclonal antibodies have molecular specificity to the extracellular domain of EGFR. The intensity of light scattering from the labeled SiHa cells is ca. 50 times higher than from unlabeled cells. Therefore unlabeled cells cannot be resolved on the dark background. No labeling was observed when gold conjugates with nonspecific mouse monoclonal IgG antibodies were added to the cells. We estimated the average amount of gold conjugates bound per cell. We centrifuged the labeled cells and measured the decrease in optical density of the supernatant relative to the original suspension of the conjugates. We calculated approximately 5×10⁴ conjugates are bound per cell, which correlates well with previously reports that most cell types express from 2×10⁴ to 20×10⁴ EGF receptors per cell (81).


Figure 10: Laser scanning confocal reflectance (A) and combined confocal reflectance/transmittance (B) images of labeled SiHa cells. Scattering from gold conjugates is false-colored in red. The confocal reflectance and transmittance images were obtained independently and then overlaid. The scale bar is ca. 20 mm.

Light scattering from the labeled cells is so strong that it can be easily observed using low magnification optics and an inexpensive light source such as a laser pointer. Figure 11 shows a series of images of labeled SiHa cells placed on a microscope slide obtained using a 20× objective. In bright-field transmission, the cells with bound gold conjugates appear darker due to light absorption by the metal nanoparticles and the unlabeled cells appear more transparent (Fig. 11a). When the sample is illuminated by a laser pointer at grazing incidence, the labeled cells appear bright due to scattered light (Fig. 11b). Finally, after bright-field illumination is turned off, only labeled cells can be seen (Fig. 11c). We believe that we can easily image this strong signal using the multi-spectral digital imaging systems that we are developing. Areas which indicate the presence of contrast agent would then be probed at higher spatial resolution using confocal microscopy.


Figure 11: SiHa cells labeled with anti-EGFR gold conjugates in (a) brightfield (b) in brightfield with laser pointer illumination, and (c) with just laser pointer illumination.

We extended our work using anti-EGFR gold nanoparticle conjugates to label organ cultures of normal and neoplastic cervical tissue. Cultures were incubated in a solution containing contrast agent, washed and then imaged with reflectance based confocal microscopy. The bright "honey-comb" like structure of labeled cellular cytoplasm membranes of closely spaced cells can be easily seen in clinically abnormal samples of cervical biopsies obtained using confocal reflectance microscope (Fig. 12A). The labeling is much less pronounced in clinically normal samples. No labeling of the normal biopsy can be seen when the sample is imaged under the same acquisition conditions as the abnormal sample (Fig. 12B). Anti-EGFR/gold conjugates do not bind to the stromal layer of cervical biopsies.


Figure 12: Cervical biopsies labeled with anti-EGFR antibodies/gold nanoparticles conjugates: (A) clinically abnormal; (B) clinically normal obtained under the same acquisition conditions as (A).

In vivo application of these contrast agents depends on the ability to deliver the agents throughout the epithelium in the organ site of interest. Pre-cancers of squamous epithelium originate at the basal layer, which can be located 300-500μm beneath the tissue surface; therefore, to develop new diagnostic tools and to study the earliest molecular changes associated with cancer progression it is imperative to deliver the gold nanoparticles throughout the whole epithelium. Using engineered tissue constructs (82), we demonstrated that penetration enhancers used for topical drug delivery such as polyvinyl pyrrolidone (PVP), can be used to deliver the gold nanoparticles throughout the epithelium. DMSO and PVP have been previously studied as penetration enhancer in topical delivery of drugs. Gold bioconjugates with anti-EGFR monoclonal antibodies in different formulations were applied on top of the constructs at room temperature. Then the tissue constructs were washed in PBS and 200-micron transverse sections were prepared. Only the surface of the engineered tissue constructs is labelled when anti-EGFR gold conjugates are topically applied in PBS to the surface of the constructs (Fig. 13, top row). However, the whole depth (up to 600μm) of the constructs is uniformly labelled when the conjugates are applied in the presence of either DMSO or PVP (Fig. 13). Imaging at higher magnification using 40X objective showed that labeling is confined to plasma membrane of the cells (Fig. 13D). No labeling was observed when conjugates of gold nanoparticles with nonspecific monoclonal antibodies were used.

Metal Nanoparticles and MMP

To demonstrate the feasibility of imaging of MMPs using contrast agents based on metal nanoparticles we prepared gold conjugates with monoclonal antibodies for MMP-2 (data not shown) and MMP-9. The conjugates were used to label cervical epithelial cancer cells grown on two different substrates: a pure collagen I gel and a collagen I gel in the presence of 5% gelatin. SiHa cells were placed on a substrate and allowed to grow for 5-24 hrs in DMEM with 5% FBS at 37°C and 5% CO₂, and then antibody-gold conjugates were applied to a sample in PBS for 20-30 minutes under sterile conditions. Excess contrast agent was removed and the sample was imaged using a confocal reflectance/fluorescence microscope without intermediate washing. Reflectance images were obtained with 647 nm excitation and fluorescence was excited at 360 nm and collected using 405 band-pass emission filter.

Figure 14 shows the results of labeling of SiHa cells grown on collagen I substrate with anti-MMP-9/gold conjugates. Significant labeling of cellular cytoplasm was observed. We suggest that cytoplasmic labeling is associated with internalization of labeled MMP-9 molecules from the plasma membrane. Rapid internalization and degradation of MMPs including MMP-9 (83) is an important mechanism in regulating extracellular proteinase activity. We also observed strong labeling of collagen fibers located along clusters of cells (Fig. 14A and C). Previously, similar labeling was observed using fluorescent labeled antibodies in fixed 3-D tissue cultures of cells inside collagen I matrix (84). It was attributed to membrane deposits which are shed by migrating cells along their tracks in collagen matrix. These deposits contain a variety of plasma membrane proteins including MMPs. Our images show a number of elongated polarized cells, indicative of cellular migration. Little cell or ECM labeling was observed in areas with low cell density.


Figure 14: Confocal reflectance (A and C) and fluorescence (B and D) images of SiHa cells on collagen I labeled with anti-MMP-9/gold conjugates. The area in the white square in (A) is shown at higher magnification in (C). Arrows show polarized cells.

Metal Nanoparticles and Intracellular Targets

Figure 15 shows preliminary results obtained labeling SiHa cells with 10 nm gold nanoparticles conjugated to anti-E7 monoclonal antibodies. Cells were incubated in contrast agent in PBS and in PBS with 10% PVP. Following incubation, cells were washed and imaged using laser scanning confocal microscopy. Figure 15 shows the co-localized autofluorescence (green) and reflectance (white) images from SiHa cells incubated with PVP. Autofluorescence is limited to the cytoplasm, whereas backscattering is produced by nanoparticles within the nucleus. No backscattering was observed in cells incubated with contrast agent in PBS alone. While extremely preliminary, these results indicate that delivery and detection of contrast agent is feasible. In preliminary data we observed at least a three fold, statistically significant increase in nuclear backscattering from nanoparticles in cells with high E7 expression compared to cells with low E7 expression.


Figure 15: Co-localized fluorescence (green) and reflectance (white) laser scanning confocal microscopic images obtained from SiHa cells incubated in anti-E7 gold nanoparticle conjugates with 10% PVP. Autofluorescence due to NAD(P)H is observed in the cytoplasm, while strong backscattering due to contrast agents is seen in the nucleus.

Contrast agents based on gold nanoparticle antibody conjugates have the potential for in vivo use, with topical or systemic delivery. The inherent biocompatibility of gold implies they can be used directly in vivo without the need for protective layer growth. In fact, long term treatment of rheumatoid arthritis utilizes gold (85) (up to a cumulative dose of 1.2-1.8 g/year for up to 10 years). We anticipate that less than 0.3 mg of gold would be required for diagnosis with topical delivery to the cervix. Humanized antibodies, where a mouse antibody-binding site is transferred to a human antibody gene, are much less immunogenic in humans (86), and many humanized antibodies are currently in clinical trials. Since 1997, the FDA has approved more than 10 monoclonal antibody based drugs, including Herceptin for metastatic breast cancer therapy (87, 88). For surface lesions located in epithelial tissue, simple FDA approved agents, such as polyvinylpyrrolidone can be used to increase tissue permeability and deliver contrast agents topically.

Organic Fluorescent Dyes and EGFR

Fluorescence imaging is one of the oldest and the most popular molecular imaging techniques. A variety of molecular contrast agents and beacons have been developed for fluorescence microscopic imaging. For example conjugates of a near infrared fluorescent dye to antibodies against EGFR have shown promise for detection of precancers (89). Successful tumor-specific targeting using organic dye-peptide conjugates was demonstrated in animal models (90, 91, 92, 93, 94). Activatable fluorescence imaging probes to sense enzyme activity have been used to image protease activity in tumors (95, 96). The processes of tumor metastasis and angiogenesis have been studied in live animals using cancer cell lines expressing green fluorescent protein (GFP) (32, 97). Fluorescent dyes such as fluorescein (FITC) and indocyanine green (ICG) have been approved by the FDA for clinical use in human subjects. Fluorescein angiography is a standard procedure in ophthalmology and ICG has been used to study retinal and chorodial circulation for more than 30 years (98, 100). Recently, it was also shown that ICG is a promising fluorescent dye for cancer detection (101). Molecular specific imaging using fluorescein labeled antibodies (102) and indocyanine green N-hydroxysulfosuccinimide ester (ICG-sulfo-Osu) labeled antibodies (103) was also demonstrated.

In our initial experiments we are exploring imaging EGFR in cervical cancer and oral cavity cell lines, 3D tissue cultures, and organ cultures using fluorescent dyes conjugated to antibody against EGFR. The labeling was performed in a two-step process by first incubating the specimens with a biotinylated antibody against EGFR and then with Alexa Fluor® 660 streptavidin. All samples were first blocked to prevent binding to endogenous biotin by incubating for approximately 15 minutes with an avidin solution followed by a biotin solution. We demonstrated that labeling is confined to plasma membrane of oral cavity and cervical cells. We also showed that PVP solution can be used to allow topical delivery of the contrast agents through multiple layers of epithelial cells. Similar to experiments with gold nanoparticles a "honey-comb" like pattern due to labeling of cellular plasma membrane was seen in abnormal biopsies from oral cavity (Fig. 16). Significant contrast was observed between labeled normal and abnormal oral cavity.


Figure 16: Tissue slices from oral cavity biopsies labeled with anti-EGFR antibodies conjugated to Alexa Fluor® 660 streptavidin: clinically abnormal (left) and clinically normal (right).

Quantum Dots and EGFR

A variety of semiconductor nanocrystals with characteristic lengths typically on the order of 1-10 nm were named quantum dots. Many interesting properties of quantum dots result from quantum-size confinement including their luminescence. Fluorescence emission of quantum dots is size dependable and can range from 400 nm to 2μm with very narrow typical emission width of approximately 20-30 nm (104, 105). Quantum dots of different sizes that emit fluorescence at different wavelength all can be excited at a single wavelength at the same time. This provides a unique opportunity to do multi-color imaging experiments with a single excitation wavelength (106). Despite the obvious advantages of quantum dots as compared to conventionally used fluorescence labels, their biological applications have been hampered by the low solubility of semiconductor materials which comprise the dots. Recently, new chemical strategies were proposed to make water soluble quantum dots that immediately resulted in applications of quantum dots for biological imaging (107, 108, 109, 110, 111, 112). Comparison of quantum dots to one of the brightest fluorescent molecules -- rhodamine 6G (R6G) -- showed that the quantum dots are 20 times as bright, 100 times as stable against photobleaching, and one-third as wide in spectral linewidth (112).

More recently, the first in vivo applications of quantum dots were demonstrated (109, 110). In (108) quantum dots conjugated with peptides specific for normal lung or tumor blood vessels, or for tumor lymphatic vessels were i.v.-injected into tumor bearing mice. Specific targeting of lung or tumor vasculature using peptide-coated quantum dots was demonstrated. No acute toxicity associated with i.v. administration of the quantum dots was observed even after 24 hours of circulation. In another study (110) quantum dots were solubilized for biological in vivo imaging by encapsulation inside phospholipid block-copolymer micelles. The encapsulated dots were microinjected into individual cells at early-stage Xenopus embryos. The authors followed development of the labeled cells because the dots were confined to progeny of the injected cells. The encapsulated quantum dots do not exhibit any biological activity and are non-toxic for embryos at the levels of ca. 2 × 10⁹ dots/cell. These studies suggest that the labeled nanoparticles are stable in the in vivo environment.

We have also begun studies to image cells and fresh tissue slices with quantum dots. SiHa cervical cancer cells were incubated with a biotinylated anti-EGFR monoclonal antibody (clone 111.6, LabVision). As controls, cells were incubated with biotinylated normal mouse IgG or with PBS only. A 10 nM solution of the 605 nm quantum dot streptavidin conjugate (Quantum Dot Corp.) was prepared and cells were incubated for 1 hour. As an additional control, cells which had not been exposed to antibody or the quantum dot solution were also prepared. After final washing, imaging was performed on a laser scanning confocal microscope with 488 nm and 568 nm excitation. A fresh abnormal biopsy from the floor of the mouth of a consenting patient was obtained from the MD Anderson Cancer Center and maintained in tissue media. The biopsy was sliced into 200 micron transverse sections. Before labeling, the tissue slice was blocked for endogenous biotin using streptavidin. Labeling and imaging the tissue slice proceeded as above. Results (Fig. 17) show labeling of the cervical cancer cells and the abnormal tissue slice; control images showed significantly less intensity. Images at the two excitation wavelengths both yield bright signal, illustrating the broad excitation maxima, which simplifies multi-target, multi-color imaging.


Figure 17: Top row: Confocal fluorescence images of SiHa cells labeled with anti-EGFR quantum dots at 488 nm excitation (left) and 568 nm excitation (right). The bottom left photo shows the same field in transmission mode. The bottom right image shows a fresh tissue slice of abnormal oral cavity labeled with the anti-EGFR quantum dots at 488 nm excitation. In all cases, membrane labeling is observed.

Discussion

This paper summarizes developments in optical imaging and contrast agents that have the potential to image the molecular features of cancer in real time, in vivo. While these technologies have been used successfully in the laboratory and in limited human and animal trials, there are important challenges to be addressed before these approaches can be translated successfully to clinical practice. First, cost-effective imaging systems which can yield images with sufficient signal to noise ratio and sub-cellular spatial resolution must be developed. While a number of such systems have been developed and tested, there are engineering challenges associated with producing inexpensive systems that are easy to align and maintain in the clinical environment. Furthermore, it is important to develop optical systems with "zoom" capability so that large areas of tissue at risk can be quickly interrogated for suspicious areas and these areas can then be examined in more detail with higher spatial resolution. Image registration and stabilization will be important challenges to address in this development.

In order to use such optical systems to image molecular features of cancer, it will be necessary to deliver sufficient contrast agent to tissue that a reasonable signal to noise ratio can be obtained. In the initial steps of the development the results of optical imaging must be validated using standard histopathology and immunohistochemistry. While preliminary animal studies indicate that optical molecular imaging of cancer biomarkers in vivo is feasible, it will be important to carefully evaluate the tradeoffs between detectability and toxicity. In particular, strategies must be developed to adequately deliver contrast agent to deeper structures and intracellular targets. The ability to deliver contrast agent sufficiently rapidly to enable real time surgical guidance must be rigorously evaluated, taking care to examine how rapidly labeling occurs and how rapidly unbound contrast agent can be cleared. Before contrast agents can be used in human subjects, extensive animal studies must be carried out to evaluate any potential toxicity of these contrast agents and delivery formulations.

Conclusions

We believe these contrast agents and imaging systems have the potential to significantly impact current clinical practice (Fig. 18). The ability to rapidly and non-invasively image molecular features of cancer using inexpensive imaging systems can improve screening for and early detection of neoplasia, can provide more effective determination of tumor margins, and can be used to study response to treatment non-invasively. At the same time, the contrast agents have the potential to provide the ability to study the molecular processes associated with carcinogenesis in vivo in humans. In particular, they may provide the ability to directly image the biology of invasion and to study host response serially over time. In the future, the integration of optical imaging and optically-active, molecular-specific contrast agents may be used to identify lesions at high risk of progression based on both molecular and phenotypic markers. This knowledge can then potentially be used to select the most appropriate choice of therapeutic agent, and the ability to image molecular features of neoplasia can be used to provide a rapid molecular assessment of response. We believe this approach will translate to decreased incidence, morbidity, and mortality.



Figure 18: Uses for molecular-specific optical contrast agents.
References

1. Organization, W. H. Cancer Control (1996).
   
2. Frater, J. L. et al. Chronic Myeloid Leukemia Following Therapy with Imatinib Mesylate (Gleevec). Bone Marrow Histopathology and Correlation with Genetic Status. American Journal of Clinical Pathology 119, 833-841 (2003).
   
3. Yee, K. W. and Keating, A. Advances in Targeted Therapy for Chronic Myeloid Leukemia. Expert Review of Anticancer Therapy 3, 295-310 (2003).
   
4. Pomper, M. G. Molecular Imaging: An Overview. Academic Radiology 8, 1141-1153 (2001).
   
5. Rajadhyaksha, M. et al. In Vivo Confocal Scanning Laser Microscopy of Human Skin: Melanin Provides Strong Contrast. Journal of Investigative Dermatology 104, 946-952 (1995).
   
6. Rajadhyaksha, M., Anderson, R. R., and Webb, R. H. Video-rate Confocal Scanning Laser Microscope for Imaging Human Tissues In Vivo. Applied Optics 38, 2105-2115 (1999).
   
7. Rajadhyaksha, M. et al. In Vivo Confocal Scanning Laser Microscopy of Human Skin II: Advances in Instrumentation and Comparison with Histology. Journal of Investigative Dermatology, 113, 293-303 (1999).
   
8. Rajadhyaksha, M. et al. Confocal Examination of Nonmelanoma Cancers in Thick Skin Excisions to Potentially Guide Mohs Micrographic Surgery Without Frozen Histopathology. The Journal of Investigative Dermatology 117, 1137-1143 (2001).
   
9. Rajadhyaksha, M. et al. Confocal Cross-Polarized Imaging of Skin Cancers to Potentially Guide Mohs Micrographic Surgery. Optics & Photonics News, 30 (2001).
   
10. Selkin, B. et al. In Vivo Confocal Microscopy in Dermatology. Dermatologic Clinics 19, 369-377 (2001).
    
11. White, W. M. et al. Noninvasive Imaging of Human Oral Mucosa In Vivo by Confocal Reflectance Microscopy. Laryngoscope 109, 1709-1717 (1999).
    
12. González, S. et al. Confocal Reflectance Imaging of Folliculitis In Vivo: Correlation with Routine Histology. Journal of Cutaneous Pathology 26, 201-205 (1999).
    
13. González, S. et al. Allergic Contact Dermatitis: Correlation of In Vivo Confocal Imaging to Routine Histology. Journal of the American Academy of Dermatology 40, 708-713 (1999).
    
14. González, S. et al. Characterization of Psoriasis In Vivo by Reflectance Confocal Microscopy. Journal of Medicine 30, 337-56 (1999).
    
15. González, S. et al. In Vivo Abnormal Keratinazation in Darier-White's Disease as Viewed by Real-time Confocal Imaging. Journal of Cutaneous Pathology 26, 504-508 (1999).
    
16. González, S. et al. Confocal Imaging of Sebaceous Gland Hyperplasia In Vivo to Assess Efficacy and Mechanism of Pulsed Dye Laser Treatment. Lasers in Surgery and Medicine 25, 8-12 (1999).
    
17. Huzaira, M. et al. Topographic Variations in Normal Skin, as Viewed by In Vivo Reflectance Confocal Microscopy. The Journal of Investigative Dermatology 116, 846-852 (2001).
    
18. Langley, R. G. et al. Confocal Scanning Laser Microscopy of Benign and Malignant Melanocytic Skin Lesions In Vivo. Journal of the American Academy of Dermatology 45, 365-376 (2001).
    
19. Collier, T. et al. Near Real-Time Confocal Microscopy of Amelanotic Tissue: Detection of Dysplasia in Ex Vivo Cervical Tissue. Academic Radiology 9, 504-512 (2002).
    
20. Collier, T. et al. Near Real Time Confocal Microscopy of Amelanotic Tissue: Dynamics of Aceto-Whitening Enable Nuclear Segmentation. Optics Express 6, 40-48 (2000).
    
21. Collier, T. et al. Fiber-Optic Confocal Microscope for Biological Imaging. in Conference on Lasers and Electro-Optics. San Francisco, CA: Institute of Electrical and Electronics Engineers (1998).
    
22. St Croix, B. et al. Genes Expressed in Human Tumor Endothelium. Science 289, 1197-1202 (2000).
    
23. Hibi, K. et al. Advances in Brief -- Serial Analysis of Gene Expression in Non-Small Cell Lung Cancer. Cancer Research: The Official Organ of the American Association for Cancer Research 58, 5690-5694 (1998).
    
24. Lal, A. et al. Advances in Brief -- A Public Database for Gene Expression in Human Cancers. Cancer Research: The Official Organ of the American Association for Cancer Research 59, 5403-5407 (1999).
    
25. Kolonin, M., Pasqualini, R., and Arap, W. Molecular Addresses in Blood Vessels as Targets for Therapy. Current Opinion in Chemical Biology 5, 308-313 (2001).
    
26. Arap, W. et al. Targeting the Prostate for Destruction Through a Vascular Address. Proceedings of the National Academy of Sciences of the United States of America 99, 1527-1531 (2002).
    
27. Takaha, N. et al. High Mobility Group Protein I(Y): A Candidate Architectural Protein for Chromosomal Rearrangements in Prostate Cancer Cells. Cancer Res. (2002).
    
28. Condeelis, J. The Biochemistry of Animal Cell Crawling, in Motion Analysis of Living Cells. pp. 85-100. Ed., D. Soll. Wiley Liss, Inc (1988).
    
29. Bailly, M. et al. Relationship Between Arp2/3 Complex and the Barbed Ends of Actin Filaments at the Leading Edge of Carcinoma Cells after Epidermal Growth Factor Stimulation. J. Cell Biol. (1999).
    
30. Condeelis, J. S., Wyckoff, J., and Segall, J. E. Imaging of Cancer Invasion and Metastasis using Green Fluorescent Protein. European Journal of Cancer 36, 1671-1680 (2000).
    
31. Gohongi, T. et al. Tumor-host Interactions in the Gallbladder Suppress Distal Angiogenesis and Tumor Growth: Involvement of Transforming Growth Factor Beta1. Nature Medicine 5, 1203-1208 (1999).
    
32. Brown, E. B. et al. In Vivo Measurement of Gene Expression, Angiogenesis and Physiological Function in Tumors using Multiphoton Laser Scanning Microscopy. Nature Medicine 7, 864-868 (2001).
    
33. Carmeliet, P. and Jain, R. K. Angiogenesis in Cancer and other Diseases. Nature 407, 249-257 (2000).
    
34. van de Vijver, M. J. et al. A Gene-Expression Signature as a Predictor of Survival in Breast Cancer. The New England Journal of Medicine 347, 1999-2009 (2002).
    
35. Ang, K. K. et al. Impact of Epidermal Growth Factor Receptor Expression on Survival and Pattern of Relapse in Patients with Advanced Head and Neck Carcinoma. Cancer Research 62, 7350-7356 (2002).
    
36. Hanahan, D. and Weinberg R. A. The Hallmarks of Cancer. Cell 100, 57-70 (2000).
    
37. Lin, C. R. et al. Expression Cloning of Human EGF Receptor Complementary DNA: Gene Amplification and Three Related Messenger RNA Products in A431 Cells.
    
38. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W., Lee, J., Yarden, Y., Libermann, T. A. et al. Human Epidermal Growth Factor Receptor cDNA Sequence and Aberrant Expression of the Amplified Gene in A431 Epidermoid Carcinoma Cells. Nature 309, 418-425 (1984).
    
39. Xu, Y. H. et al. Human Epidermal Growth Factor Receptor cDNA is Homologous to a Variety of RNAs Overproduced in A431 Carcinoma Cells. Nature 309, 806-810 (1984).
    
40. Kersemaekers, A. M. et al. Oncogene Alterations in Carcinomas of the Uterine Cervix: Overexpression of the Epidermal Growth Factor Receptor is Associated with Poor Prognosis. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 5, 577-586 (1999).
    
41. Chang, J. L. et al. The Expression of Type I Growth Factor Receptors in the Squamous Neoplastic Changes of Uterine Cervix. Gynecologic Oncology 73, 62-71 (1999).
    
42. Nishioka, T. et al. Prognostic Significance of c-erbB-2 Protein Expression in Carcinoma of the Cervix Treated with Radiotherapy. Journal of Cancer Research and Clinical Oncology 125, 96-100 (1999).
    
43. Mathevet, P., Frappart, L., and Hittelman, W. Dysplasies du col utérin: étude des expressions des gènes Rb et p53, et corrélation avec l'activité mitotique. Gynecologie, Obstetrique & Fertilite 28, 44-50 (2000).
    
44. Shin, D. M. et al. Expression of Epidermal Growth Factor Receptor, Polyamine Levels, Ornithine Decarboxylase Activity, Micronuclei, and Transglutaminase I in a 7,12-dimethylbenz(a)anthracene-induced Hamster Buccal Pouch Carcinogenesis Model. Cancer Research 50, 2505-2510 (1990).
    
45. Todd, R. and D. T. Wong. Epidermal Growth Factor Receptor (EGFR) Biology and Human Oral Cancer. Histology and Histopathology 14, 491-500 (1999).
    
46. Averbuch, S. D. Lung Cancer Prevention: Retinoids and the Epidermal Growth Factor Receptor-a Phoenix Rising? Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 8, 1-3 (2002).
    
47. Sporn, M. B. The War on Cancer. Lancet 347, 1377-1381 (1996).
    
48. Thomas, G. T., Lewis, M. P. and Speight, P. M. Matrix Metalloproteinases and Oral Cancer. Oral Oncology 35, 227-33 (1999).
    
49. Garzetti, G. G. et al. Microinvasive Cervical Carcinoma and Cervical Intraepithelial Neoplasia: Biologic Significance and Clinical Implications of 72-kDa Metalloproteinase Immunostaining. Gynecologic Oncology 61, 197-203 (1996).
    
50. Talvensaari, A. et al. Matrix Metalloproteinase 2 Immunoreactive Protein Appears Early in Cervical Epithelial Dedifferentiation. Gynecologic Oncology 72, 306-311 (1999).
    
51. Daneri-Navarro, A. et al. Proteolytic Activity in Extracts of Invasive Cervical Carcinoma and Precursor Lesions. Biomedicine & Pharmacotherapy 49, 304-310 (1995).
    
52. McCluggage, W. G., Maxwell, P. and Bharucha, H. Immunohisto-chemical Detection of Metallothionein and MIB1 in Uterine Cervical Squamous Lesions. International Journal of Gynecological Pathology: Official Journal of the International Society of Gynecological Pathologists 17, 29-35 (1998).
    
53. Davidson, B. et al. Expression of Matrix Metalloproteinase-9 in Squamous Cell Carcinoma of the Uterine Cervix - Clinicopatho-logic Study Using Immunohistochemistry and mRNA In Situ Hybridization. Gynecologic Oncology 72, 380-386 (1999).
    
54. Sienel, W. et al. Prognostic Impact of Matrix Metalloproteinase-9 in Operable Non-small Cell Lung Cancer. International Journal of Cancer 103, 647-651 (2003).
    
55. Passlick, B. et al. Overexpression of Matrix Metalloproteinase 2 Predicts Unfavorable Outcome in Early-stage Non-small Cell Lung Cancer. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 6, 3944-3948 (2000).
    
56. Hrabec, E. et al. Activity of Type IV Collagenases (MMP-2 and MMP-9) in Primary Pulmonary Carcinomas: A Quantitative Analysis. Journal of Cancer Research and Clinical Oncology 128, 197-204 (2002).
    
57. Munoz N. HPV and Cervical Neoplasia: Review of Case-control and Cohort Studies. IARC Sci Publ 119, 251-261 (1992).
    
58. Howett, J. C. Preventing Cervical Cancer. Science 288, 1753-1755 (2000).
    
59. Klaes, R. et al. Detection of High Risk Cervical Intraepithelial Neoplasia and Cervical Cancer by Amplification of Transcripts Derived from Integrated Papillomavirus Oncogenes. Cancer Research 59, 132-136 (1999).
    
60. Alberts, B. et al. Molecular Biology of the Cell. New York: Garland Publishing (1994).
    
61. Drezek, R. A. et al. Laser Scanning Confocal Microscopy of Cervical Tissue Before and After Application of Acetic Acid. American Journal of Obstetrics and Gynecology 182, 1135-1139 (2000).
    
62. Delaney, P. M. and Harris, M. R. Fiber Optics in Confocal Microscope in Handbook of Confocal Microscopy. pp. 515-523. Ed., Pawley, J. B. Plenum Press: New York (1995).
    
63. Smithpeter, C. L. et al. Near Real Time Confocal Microscopy of Cultured Amelanotic Cells: Sources of Signal, Contrast Agents, and Limits of Contrast. Journal of Biomedical Optics 3, 429-436 (1998).
    
64. Minsky, M. Microscopy Apparatus. U. S. Patent 301467 (1961).
    
65. Liang, C. et al. Design of a High-numerical-aperture Miniature Microscope Objective for an Endoscopic Fiber Confocal Reflectance Microscope. Applied Optics 41, 4603-4610 (2002).
    
66. Sung, K. B. et al. Near Real Time In Vivo Fibre Optic Confocal Microscopy: Sub-cellular Structure Resolved. Journal of Microscopy 207, 137-145 (2002).
    
67. Sung, K.-B. et al. Fiber-Optic Confocal Reflectance Microscope With Miniature Objective for In Vivo Imaging of Human Tissues. IEEE Transactions on Biomedical Engineering 49, 1168-1172 (2002).
    
68. Hornbeck, L. J. Digital Light Processing and MEMS: Reflecting the Digital Display Needs of the Networked Society in SPIE/EOS European Symposium on Lasers, Optics, and Vision for Productivity in Manufacturing 1, Conference on Micro-Optical Tech. For Measurement, Sensors & Microsystems. Besacon, France (1996).
    
69. Lane, P. M. et al. Medical Optics and Biotechnology - Fiber-optic Confocal Microscopy using a Spatial Light Modulator. Optics Letters 25, 1780-1782 (2000).
    
70. Benavides, J. M. et al. Multispectral Digital Colposcopy for In Vivo Detection of Cervical Cancer. Optics Express 11, 1223-1236 (2003).
    
71. Horisberger, M. Colloidal Gold: A Cytochemical Marker for Light and Fluorescent Microscopy and for Transmission and Scanning Electron Microscopy. Scan Electron Microsc 2, 9-31 (1981).
    
72. Geoghegan, W. D. and Ackerman, G. A. Adsorption of Horseradish Peroxidase, Ovomucoid and Anti-immunoglobulin to Colloidal Gold for the Indirect Detection of Concanavalin A, Wheat Germ Agglutinin and Goat Anti-human Immunoglobulin G on Cell Surfaces at the Electron Microscopic Level: A New Method, Theory and Application. The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society 25, 1187-1200 (1977).
    
73. Mulvaney, P. Surface Plasmon Spectroscopy of Nanosized Metal Particles. Langmuir 12, 788-800 (1996).
    
74. Nabiev, I. R., Morjani, H. and Manfait, M. Selective Analysis of Antitumor Drug Interaction with Living Cancer Cells as Probed by Surface-enhanced Raman Spectroscopy. European Biophysics Journal 19, 311-316 (1991).
    
75. Kneipp, K. et al. Surface-Enhanced Raman Spectroscopy in Single Living Cells Using Gold Nanoparticles. Applied Spectroscopy 56, 150-154 (2002).
    
76. Nie, S. and Emory, S. R. Probing Single Molecules and Single Nanoparticles by Surface-enhanced Raman Scattering. Science 275, 1102-1106 (1997).
    
77. Elghanian, R. et al. Selective Colorimetric Detection of Polynucleotides Based on the Distance-dependent Optical Properties of Gold Nanoparticles. Science 277, 1078-1080 (1997).
    
78. Yasuda, R. et al. Resolution of Distinct Rotational Substeps by Submillisecond Kinetic Analysis of F1-ATPase. Nature 410, 898-904 (2001).
    
79. Schultz, S. et al. Single-target Molecule Detection with Nonbleaching Multicolor Optical Immunolabels. Proceedings of the National Academy of Sciences of the United States of America 97, 996-1001 (2000).
    
80. Sokolov, K. et al. Real-time Vital Optical Imaging of Precancer using Anti-epidermal Growth Factor Receptor Antibodies Conjugated to Gold Nanoparticles. Cancer Research 63, 1999-2004 (2003).
    
81. Carpenter, G. Receptors For Epidermal Growth Factor And Other Polypeptide Mitogens. Annual Review of Biochemistry 56, 881-914 (1987).
    
82. Sokolov, K. et al. Realistic Three-dimensional Epithelial Tissue Phantoms for Biomedical Optics. Journal of Biomedical Optics 7, 148-156 (2002).
    
83. Hahn-Dantona, E. et al. The Low Density Lipoprotein Receptor-related Protein Modulates Levels of Matrix Metalloproteinase 9 (MMP-9) by Mediating its Cellular Catabolism. The Journal of Biological Chemistry 276, 15498-15503 (2001).
    
84. Friedl, P., Brocker, E.-B. The Biology of Cell Locomotion within Three-dimensional Extracellular Matrix. Cellular and Molecular Life Sciences 57, 41-64 (2000).
    
85. Abrams, M. J., Murrer, B. A. Metal Compounds in Therapy and Diagnosis. Science 261, 725-730 (1993).
    
86. Berkower, I. The Promise and Pitfalls of Monoclonal Antibody Therapeutics. Current Opinion in Biotechnology 7, 622-628 (1996).
    
87. Holliger, P., Bohlen, H. Engineering Antibodies for the Clinic. Cancer and Metastasis Reviews 18, 411-419 (1999).
    
88. Weiner, L. M. An Overview of Monoclonal Antibody Therapy of Cancer. Seminars in Oncology 26, 41-50 (1999).
    
89. Soukos, N. S. et al. Epidermal Growth Factor Receptor-targeted Immunophotodiagnosis and Photoimmunotherapy of Oral Precancer In Vivo. Cancer Research 61, 4490-4496 (2001).
    
90. Achilefu, S. et al. New Approach to Optical Imaging of Tumors. Proceedings of SPIE: Biomarkers and Biological Spectral Imaging. San Jose, CA (2001).
    
91. Achilefu, S. et al. Site-specific Tumor Targeted Fluorescent Contrast Agents. Proceedings of SPIE: Clinical Lasers and Diagnostics. Amsterdam (2001).
    
92. Bugaj, J. E. et al. Novel Fluorescent Contrast Agents for Optical Imaging of In Vivo Tumors Based on a Receptor-targeted Dye-peptide Conjugate Platform. Journal of Biomedical Optics 6, 122-133 (2001).
    
93. Tung, C. H. et al. A Receptor-targeted Near-infrared Fluorescence probe for In Vivo Tumor Imaging. Chembiochem 3, 784-786 (2002).
    
94. Becker, A. et al. Receptor-targeted Optical Imaging of Tumors with Near-infrared Fluorescent Ligands. Nature Biotechnology 19, 327-31 (2001).
    
95. Bremer, C., Tung, C. H. and Weissleder, R. In Vivo Molecular Target Assessment of Matrix Metalloproteinase Inhibition. Nature Medicine 7, 743-748 (2001).
    
96. Tung, C. H. et al. In Vivo Imaging of Proteolytic Enzyme Activity using a Novel Molecular Reporter. Cancer Research 60, 4953-4958 (2000).
    
97. Farina, K. L. et al. Cell Motility of Tumor Cells Visualized in Living Intact Primary Tumors using Green Fluorescent Protein. Cancer Research 58, 2528-2532 (1998).
    
98. Bornhop, D. J. et al. Advance in Contrast Agents, Reporters, and Detection. Journal of Biomedical Optics 6, 106-110 (2001).
    
99. Desmettre, T., Devoisselle, J. M. and Mordon, S. Fluorescence Properties and Metabolic Features of Indocyanine Green (ICG) as Related to Angiography. Survey of Ophthalmology 45, 15-27 (2000).
    
100. Slakter, J. S. et al. Indocyanine-green Angiography. Current Opinion in Ophthalmology 6, 25-32 (1995).
     
101. Haglund, M. M., Berger, M. S. and Hochman, D. W. Enhanced Optical Imaging of Human Gliomas and Tumor Margins. Neurosurgery 38, 308-317 (1996).
     
102. Anikijenko, P. et al. In Vivo Detection of Small Subsurface Melanomas in Athymic Mice using Noninvasive Fiber Optic Confocal Imaging. The Journal of Investigative Dermatology 117, 1442-1448 (2001).
     
103. Ito, S. et al. Detection of Human Gastric Cancer in Resected Specimens using a Novel Infrared Fluorescent Anti-human Carcinoembryonic Antigen Antibody with an Infrared Fluorescence Endoscope In Vitro. Endoscopy 33, 849-53 (2001).
     
104. Banin, U. et al. Size-dependent Electronic Level Structure of InAs Nanocrystal Quantum Dots: Test of Multiband Effective Mass Theory. Journal of Chemical Physics 109, 2306 (1998).
     
105. Murray, C. B., Norris, D. J. and Bawendi, M. G. Synthesis and Characterization of Nearly Monodisperse CdE (E = Sulfur, Selenium, Tellurium) Semiconductor Nanocrystallites. Journal of the American Chemical Society 115, 8706-8715 (1993).
     
106. Lacoste, T. D. et al. Ultrahigh-resolution Multicolor Colocalization of Single Fluorescent Probes. Proceedings of the National Academy of Sciences of the United States of America 97, 9461-6 (2000).
     
107. Chan, W. C. et al. Luminescent Quantum Dots for Multiplexed Biological Detection and Imaging. Current Opinion in Biotechnology 13, 40-46 (2002).
     
108. Parak, W. J. et al. Cell Motility and Metastatic Potential Studies based on Quantum Dot Imaging of Phagokinetic Tracks. Advanced Materials 14, 882-885 (2002).
     
109. Akerman, M. E. et al. Nanocrystal Targeting In Vivo. Proceedings of the National Academy of Sciences of the United States of America 99, 12617-12621 (2002).
     
110. Dubertret, B. et al. In Vivo Imaging of Quantum Dots Encapsulated in Phospholipid Micelles. Science 298, 1759-1762 (2002).
     
111. Bruchez, M. Jr. et al. Semiconductor Nanocrystals as Fluorescent Biological Labels. Science 281, 2013-2016 (1998).
     
112. Chan, W. C. and Nie, S. Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science 281, 2016-8 (1998).