Alterations in the mir-15a/16-1 Loci Impairs Its Processing and Augments B-1 Expansion in De Novo Mouse Model of Chronic Lymphocytic Leukemia (CLL).

New Zealand Black (NZB) mice, a de novo model of CLL, share multiple characteristics with CLL patients, including decreased expression of miR-15a/16-1. We previously discovered a point mutation and deletion in the 3' flanking region of mir-16-1 of NZB and a similar mutation has been found in a small number of CLL patients. However, it was unknown whether the mutation is the cause for the reduced miR-15a/16-1 expression and CLL development. Using PCR and in vitro microRNA processing assays, we found that the NZB sequence alterations in the mir-15a/16-1 loci result in deficient processing of the precursor forms of miR-15a/16-1, in particular, we observe impaired conversion of pri-miR-15a/16-1 to pre-miR-15a/16-1. The in vitro data was further supported by derivation of congenic strains with replaced mir-15a/16-1 loci at one or both alleles: NZB congenic mice (NmiR+/-) and DBA congenic mice (DmiR-/-). The level of miR-15a/16-1 reflected the configuration of the mir-15a/16-1 loci with DBA congenic mice (DmiR-/-) showing reduced miR-15a levels compared to homozygous wild-type allele, while the NZB congenic mice (NmiR+/-) showed an increase in miR-15a levels relative to homozygous mutant allele. Similar to Monoclonal B-cell Lymphocytosis (MBL), the precursor stage of the human disease, an overall expansion of the B-1 population was observed in DBA congenic mice (DmiR-/-) relative to wild-type (DmiR+/+). These studies support our hypothesis that the mutations in the mir-15a/16-1 loci are responsible for decreased expression of this regulatory microRNA leading to B-1 expansion and CLL development.

Grazing incidence angle based sensing approach integrated with fiber-optic Fourier transform infrared (FO-FTIR) spectroscopy for remote and label-free detection of medical device contaminations.

Contamination of medical devices has become a critical and prevalent public health safety concern since medical devices are being increasingly used in clinical practices for diagnostics, therapeutics and medical implants. The development of effective sensing methods for real-time detection of pathogenic contamination is needed to prevent and reduce the spread of infections to patients and the healthcare community. In this study, a hollow-core fiber-optic Fourier transform infrared spectroscopy methodology employing a grazing incidence angle based sensing approach (FO-FTIR-GIA) was developed for detection of various biochemical contaminants on medical device surfaces. We demonstrated the sensitivity of FO-FTIR-GIA sensing approach for non-contact and label-free detection of contaminants such as lipopolysaccharide from various surface materials relevant to medical device. The proposed sensing system can detect at a minimum loading concentration of approximately 0.7 µg/cm(2). The FO-FTIR-GIA has the potential for the detection of unwanted pathogen in real time.

In vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment.

PURPOSE: Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. EXPERIMENTAL DESIGN: HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. RESULTS: Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. CONCLUSIONS: The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients.

In vivo assessment of HER2 receptor density in HER2-positive tumors by near-infrared imaging, using repeated injections of the fluorescent probe.

HER2 overexpression and amplification of the HER2/neu gene have been found in approximately 25% of invasive breast carcinomas. They are associated with a poor prognosis and resistance to therapy in breast cancer patients. Up to now, clinical evaluation of human epidermal growth factor receptor 2 (HER2) expression is based on ex vivo methods (immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) staining of biopsied tissue). Our goal is to realize "image and treat" paradigm using targeted fluorescent probes to evaluate expression levels of cell biomarkers responsible for cancer progression and to monitor the efficacy of corresponding monoclonal antibody treatments. We used fluorescent Affibody-based probes for in vivo analysis of HER2 receptors using near-infrared optical imaging that do not interfere with binding of the therapeutic agents to these receptors. We have analyzed two types of breast carcinoma xenografts with significant differences in HER2 expression (31 and 21 according to classification) in the mouse model. Using our kinetic model to analyze the temporal variations of the fluorescence intensity in the tumor area after two subsequent injections allowed us to assess quantitatively the difference in HER2 expression levels for two tumor types (BT-474 and MD-MBA-361). This result was substantiated by ELISA ex vivo assays of HER2 expression in the same tumors.

Fiber-optic Fourier transform infrared spectroscopy for remote label-free sensing of medical device surface contamination.

As a potential major source of biochemical contamination, medical device surfaces are of critical safety concerns in the clinical practice and public health. The development of innovative sensing methods for accurate and real-time detection of medical device surface contamination is essential to protect patients from high risk infection. In this paper, we demonstrate an alternative fiber-optic Fourier Transform Infrared (FTIR) spectroscopy based sensing approach for remote, non-contact, and label-free detection of biochemical contaminants in the mid-infrared (mid-IR) region. The sensing probe is designed using mid-IR hollow fibers and FTIR measurements are carried out in reflection mode. Bovine Serum Albumin (BSA) and bacterial endotoxin of different concentrations under thoroughly dry condition are used to evaluate the detection sensitivity. The devised system can identify ≤0.0025% (≤4 × 10(11) molecules) BSA and 0.5% (0.5 EU/ml) endotoxin concentration. The developed sensing approach may be applied to detect various pathogens that pose public health threats.

Evaluation of non-invasive multispectral imaging as a tool for measuring the effect of systemic therapy in Kaposi sarcoma.

Diffuse multi-spectral imaging has been evaluated as a potential non-invasive marker of tumor response. Multi-spectral images of Kaposi sarcoma skin lesions were taken over the course of treatment, and blood volume and oxygenation concentration maps were obtained through principal component analysis (PCA) of the data. These images were compared with clinical and pathological responses determined by conventional means. We demonstrate that cutaneous lesions have increased blood volume concentration and that changes in this parameter are a reliable indicator of treatment efficacy, differentiating responders and non-responders. Blood volume decreased by at least 20% in all lesions that responded by clinical criteria and increased in the two lesions that did not respond clinically. Responses as assessed by multi-spectral imaging also generally correlated with overall patient clinical response assessment, were often detectable earlier in the course of therapy, and are less subject to observer variability than conventional clinical assessment. Tissue oxygenation was more variable, with lesions often showing decreased oxygenation in the center surrounded by a zone of increased oxygenation. This technique could potentially be a clinically useful supplement to existing response assessment in KS, providing an early, quantitative, and non-invasive marker of treatment effect.

Affibody-DyLight conjugates for in vivo assessment of HER2 expression by near-infrared optical imaging.

PURPOSE: Amplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression in vivo would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for in vivo evaluation of the receptor status by near-infrared (NIR) optical imaging. EXPERIMENTAL DESIGN: Affibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested in vitro. For in vivo validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by ex vivo analysis. RESULTS: Affibody-DyLight conjugates showed high affinity to HER2 (K(D) = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue. CONCLUSIONS: The results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.

In vivo method to monitor changes in HER2 expression using near-infrared fluorescence imaging.

Human epidermal growth factor receptor type 2 (HER2) is a well-known biomarker that is overexpressed in many breast carcinomas. HER2 expression level is an important factor to optimize the therapeutic strategy and monitor the treatment. We used albumin binding domain-fused HER2-specific Affibody molecules, labeled with Alexa Fluor750 dye, to characterize HER2 expression in vivo. Near-infrared optical imaging studies were carried out using mice with subcutaneous HER2-positive tumors. Animals were divided into groups of five: no treatment and 12 hours and 1 week after treatment of the tumors with the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). The compartmental ligands-receptor model, describing binding kinetics, was used to evaluate HER2 expression from the time sequence of the fluorescence images after the intravenous probe injection. The normalized rate of accumulation of the specific fluorescent biomarkers, estimated from this time sequence, linearly correlates with the conventional ex vivo enzyme-linked immunosorbent assay (ELISA) readings for the same tumor. Such correspondence makes properly arranged fluorescence imaging an excellent candidate for estimating HER2 overexpression in tumors, complementing ELISA and other ex vivo assays. Application of this method to the fluorescence data from HER2-positive xenografts reveals that the 17-DMAG treatment results in downregulation of HER2. Application of the AngioSense 750 probe confirmed the antiangiogenic effect of 17-DMAG found with Affibody-Alexa Fluor 750 conjugate.

In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

HER2-affitoxin: a potent therapeutic agent for the treatment of HER2-overexpressing tumors.

PURPOSE: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2. EXPERIMENTAL DESIGN: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays. RESULTS: In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T(1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose. CONCLUSIONS: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies.

Quantitative principal component model for skin chromophore mapping using multi-spectral images and spatial priors.

We describe a novel reconstruction algorithm based on Principal Component Analysis (PCA) applied to multi-spectral imaging data. Using numerical phantoms, based on a two layered skin model developed previously, we found analytical expressions, which convert qualitative PCA results into quantitative blood volume and oxygenation values, assuming the epidermal thickness to be known. We also evaluate the limits of accuracy of this method when the value of the epidermal thickness is not known. We show that blood volume can reliably be extracted (less than 6% error) even if the assumed thickness deviates 0.04mm from the actual value, whereas the error in blood oxygenation can be as large as 25% for the same deviation in thickness. This PCA based reconstruction was found to extract blood volume and blood oxygenation with less than 8% error, if the underlying structure is known. We then apply the method to in vivo multi-spectral images from a healthy volunteer's lower forearm, complemented by images of the same area using Optical Coherence Tomography (OCT) for measuring the epidermal thickness. Reconstruction of the imaging results using a two layered analytical skin model was compared to PCA based reconstruction results. A point wise correlation was found, showing the proof of principle of using PCA based reconstruction for blood volume and oxygenation extraction.

A CTRW-based model of time-resolved fluorescence lifetime imaging in a turbid medium.

We develop an analytic model of time-resolved fluorescent imaging of photons migrating through a semi-infinite turbid medium bounded by an infinite plane in the presence of a single stationary point fluorophore embedded in the medium. In contrast to earlier models of fluorescent imaging in which photon motion is assumed to be some form of continuous diffusion process, the present analysis is based on a continuous-time random walk (CTRW) on a simple cubic lattice, the object being to estimate the position and lifetime of the fluorophore. Such information can provide information related to local variations in pH and temperature with potential medical significance. Aspects of the theory were tested using time-resolved measurements of the fluorescence from small inclusions inside tissue-like phantoms. The experimental results were found to be in good agreement with theoretical predictions provided that the fluorophore was not located too close to the planar boundary, a common problem in many diffusive systems.

Principal component model of multispectral data for near real-time skin chromophore mapping.

Multispectral images of skin contain information on the spatial distribution of biological chromophores, such as blood and melanin. From this, parameters such as blood volume and blood oxygenation can be retrieved using reconstruction algorithms. Most such approaches use some form of pixelwise or volumetric reconstruction code. We explore the use of principal component analysis (PCA) of multispectral images to access blood volume and blood oxygenation in near real time. We present data from healthy volunteers under arterial occlusion of the forearm, experiencing ischemia and reactive hyperemia. Using a two-layered analytical skin model, we show reconstruction results of blood volume and oxygenation and compare it to the results obtained from our new spectral analysis based on PCA. We demonstrate that PCA applied to multispectral images gives near equivalent results for skin chromophore mapping and quantification with the advantage of being three orders of magnitude faster than the reconstruction algorithm.

Direct curvature correction for noncontact imaging modalities applied to multispectral imaging.

Noncontact optical imaging of curved objects can result in strong artifacts due to the object's shape, leading to curvature biased intensity distributions. This artifact can mask variations due to the object's optical properties, and makes reconstruction of optical/physiological properties difficult. In this work we demonstrate a curvature correction method that removes this artifact and recovers the underlying data, without the necessity of measuring the object's shape. This method is applicable to many optical imaging modalities that suffer from shape-based intensity biases. By separating the spatially varying data (e.g., physiological changes) from the background signal (dc component), we show that the curvature can be extracted by either averaging or fitting the rows and columns of the images. Numerical simulations show that our method is equivalent to directly removing the curvature, when the object's shape is known, and accurately recovers the underlying data. Experiments on phantoms validate the numerical results and show that for a given image with 16.5% error due to curvature, the method reduces that error to 1.2%. Finally, diffuse multispectral images are acquired on forearms in vivo. We demonstrate the enhancement in image quality on intensity images, and consequently on reconstruction results of blood volume and oxygenation distributions.

Quantitative analysis of Her2 receptor expression in vivo by near-infrared optical imaging.

Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancers is associated with poor prognosis and resistance to therapy. Current techniques for estimating this important characteristic use ex vivo assays that require tissue biopsies. We suggest a novel noninvasive method to characterize HER2 expression in vivo, using optical imaging, based on HER2-specific probes (albumin-binding domain-fused-(ZHER2:342)2-Cys Affibody molecules [Affibody AB, Solna, Sweden], labeled with Alexa Fluor 750 [Molecular Probes, Invitrogen, Carlsbad, CA]) that could be used concomitantly with HER2-targeted therapy. Subcutaneous tumor xenografts, expressing different levels of HER2, were imaged with a near-infrared fluorescence small-animal imaging system at several times postinjection of the probe. The compartmental ligand-receptor model was used to calculate HER2 expression from imaging data. Correlation between HER2 amplification/overexpression in tumor cells and parameters, directly estimated from the sequence of optical images, was observed (eg, experimental data for BT474 xenografts indicate that initial slope, characterizing the temporal dependence of the fluorescence intensity detected in the tumor, linearly depends on the HER2 expression, as measured ex vivo by an enzyme-linked immunosorbent assay for the same tumor). The results obtained from tumors expressing different levels of HER2 substantiate a similar relationship between the initial slope and HER2 amplification/overexpression. This work shows that optical imaging, combined with mathematical modeling, allows noninvasive monitoring of HER2 expression in vivo.

Fluorescence lifetime imaging of activatable target specific molecular probes.

In vivo optical imaging using fluorescently labeled self-quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target-to-background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self-quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2(+) and BALB/3T3 (HER2(-)) cell lines. Changes in fluorescence lifetime correlated with temperature- and time-dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2(+) and BALB/3T3 (HER2(-))] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target-specific activatable antibody-fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging.

Affibody molecules for in vivo characterization of HER2-positive tumors by near-infrared imaging.

PURPOSE: HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. We are developing molecular probes for in vivo quantitative imaging of HER2 receptors using near-infrared (NIR) optical imaging. The goal is to provide probes that will minimally interfere with the studied system, that is, whose binding does not interfere with the binding of the therapeutic agents and whose effect on the target cells is minimal. EXPERIMENTAL DESIGN: We used three different types of HER2-specific Affibody molecules [monomer ZHER2:342, dimer (ZHER2:477)2, and albumin-binding domain-fused-(ZHER2:342)2] as targeting agents and labeled them with Alexa Fluor dyes. Trastuzumab was also conjugated, using commercially available kits, as a standard control. The resulting conjugates were characterized in vitro by toxicity assays, Biacore affinity measurements, flow cytometry, and confocal microscopy. Semiquantitative in vivo NIR optical imaging studies were carried out using mice with s.c. xenografts of HER2-positive tumors. RESULTS: The HER2-specific Affibody molecules were not toxic to HER2-overexpressing cells and their binding to HER2 did interfere with neither binding nor effectives of trastuzumab. The binding affinities and specificities of the Affibody-Alexa Fluor fluorescent conjugates to HER2 were unchanged or minimally affected by the modifications. Pharmacokinetics and biodistribution studies showed the albumin-binding domain-fused-(ZHER2:342)2-Alexa Fluor 750 conjugate to be an optimal probe for optical imaging of HER2 in vivo. CONCLUSION: Our results suggest that Affibody-Alexa Fluor conjugates may be used as a specific NIR probe for the noninvasive semiquantitative imaging of HER2 expression in vivo.

Use of scaling relations to extract intrinsic fluorescence lifetime of targets embedded in turbid media.

We present a novel method for estimating the intrinsic fluorescence lifetime of deeply embedded localized fluorophores. It is based on scaling relations, characteristic for turbid media. The approach is experimentally substantiated by successfully reconstructing lifetimes for targets at depths up to 14.5 mm. A derived correction factor was determined from the product of the transport-corrected scattering coefficient mu(s) (') and the index of refraction n(r). In addition, data from an array of detectors (> or =2) can be used to estimate mu(s) (')n(r). The suggested algorithm is a promising tool for diagnostic fluorescence, since lifetime can be a sensitive indicator of the fluorophore environment.

Using noninvasive multispectral imaging to quantitatively assess tissue vasculature.

This research describes a noninvasive, noncontact method used to quantitatively analyze the functional characteristics of tissue. Multispectral images collected at several near-infrared wavelengths are input into a mathematical optical skin model that considers the contributions from different analytes in the epidermis and dermis skin layers. Through a reconstruction algorithm, we can quantify the percent of blood in a given area of tissue and the fraction of that blood that is oxygenated. Imaging normal tissue confirms previously reported values for the percent of blood in tissue and the percent of blood that is oxygenated in tissue and surrounding vasculature, for the normal state and when ischemia is induced. This methodology has been applied to assess vascular Kaposi's sarcoma lesions and the surrounding tissue before and during experimental therapies. The multispectral imaging technique has been combined with laser Doppler imaging to gain additional information. Results indicate that these techniques are able to provide quantitative and functional information about tissue changes during experimental drug therapy and investigate progression of disease before changes are visibly apparent, suggesting a potential for them to be used as complementary imaging techniques to clinical assessment.

Fluorescence lifetime imaging system for in vivo studies.

In this article, a fluorescence lifetime imaging system for small animals is presented. Data were collected by scanning a region of interest with a measurement head, a linear fiber array with fixed separations between a single source fiber and several detection fibers. The goal was to localize tumors and monitor their progression using specific fluorescent markers. We chose a near-infrared contrast agent, Alexa Fluor 750 (Invitrogen Corp., Carlsbad, CA). Preliminary results show that the fluorescence lifetime for this dye was sensitive to the immediate environment of the fluorophore (in particular, pH), making it a promising candidate for reporting physiologic changes around a fluorophore. To quantify the intrinsic lifetime of deeply embedded fluorophores, we performed phantom experiments to investigate the contribution of photon migration effects on observed lifetime by calculating the fluorescence intensity decay time. A previously proposed theoretical model of migration, based on random walk theory, is also substantiated by new experimental data. The developed experimental system has been used for in vivo mouse imaging with Alexa Fluor 750 contrast agent conjugated to tumor-specific antibodies (trastuzumab [Herceptin]). Three-dimensional mapping of the fluorescence lifetime indicates lower lifetime values in superficial breast cancer tumors in mice.