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PDF file - 1178KB, Difference between fluorescence lifetimes at the tumor and contralateral sites (ns) vs. (a) tumor size (b) tumor depth, 6 hours after injection of Affibody probe.

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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. Clin Cancer Res; 20(13); 3531–9. ©2014 AACR.

PDF file - 1428KB, (a) Normalized fluorescence signal, measured at the tumor site, along with the fitting line, used for lifetime calculations. (b) Results of the convolution simulations to evaluate potential effect of the impulse response function (IRF) of the measurement system in the case of convolution of two exponentially decaying signals with slopes, corresponding to the IRF width and ABD-HER2-Affibody-AlexaFluor750 conjugate.

PDF file - 232KB, Average fluorescence intensity at the tumor and contralateral site (for one mouse) 12 hours after the last treatment.

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 (T1/2 < 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. Clin Cancer Res; 17(15); 5071–81. ©2011 AACR.

Infectious diseases have become one of the leading causes of global morbidity and mortality in human life. Therefore, bacteria identification plays a critical role to protect public health from infectious diseases. Optical spectroscopy has been widely used to identify bacteria by yielding unique signature spectra representing the structure and composition of the bacteria. Since bacteria exhibit very close sensing and analytical patterns, it is challenging to determine their distinctions. In this study, we introduce a novel integrative computational approach for analyzing infrared (IR) spectroscopy spectra to identify bacteria by measuring their similarities employing logarithmic-based wavelet features. A visual analytics tool is also developed to support an interactive visual analysis for characterizing bacteria features. Thirty-two signature IR spectra of Escherichia coli ( E. coli ) and forty spectra of Pseudomonas aeruginosa ( P. aeruginosai ) collected by an advanced fiber-optic Fourier transform IR (FO-FTIR) spectroscopy sensor system are used for validating the proposed computational approach. Three machine learning techniques are employed to differentiate the bacteria by measuring their performances. Implementing the presented approach, we identified the overall accuracy of 92.5% in classifying the bacteria with logistic regression. We also found that both sensitivity and specificity are significantly high, exceeding 92%.

A label-free non-contact test methodology utilizing fiber-optic-based Fourier Transform Infrared (FTIR) spectroscopy has been developed to identify potential biomarkers in cellular systems, which could be associated with disease processes or therapeutic effectiveness. As a biological in-vitro model, different metabolically active cell lines were considered in this study. FTIR absorption spectra of aggressive human glioblastoma (brain cancer) and human melanoma cell lines were compared to normal human skin fibroblasts. A ∼25-cm−1 shift in the absorption peak was observed between normal (∼1153 cm−1) and malignant cell lines (∼1177 cm−1). Furthermore, the addition of 30% H2O2 to normal skin cells produced a similar 25-cm−1 shift in the 1153-cm−1 absorption peak from the normal cells to the 1177 cm−1 found in the malignant cells, thus implicating the presence of large endogenous levels of oxidizers such as H2O2 within the cancer cells which could be responsible in producing this particular peak shift. The observed differences in the absorption spectra between normal and cancer cell lines could potentially be used to identify biomarkers within the absorption wavenumber range of 900–1300 cm−1 (7.7–11.1 μm wavelength range).

Ophthalmic viscosurgical devices (OVDs) are essential medical tools for ophthalmic surgeons to use routinely in cataract surgery. OVD endotoxin contamination has been implicated in toxic anterior segment syndrome, a severe inflammatory condition after surgery. Current standard methods for endotoxin detection in medical devices rely on dwindling Horseshoe Crab resources for Limulus amoebocyte lysate assay or rabbit intracameral and intravitreal assays. Endotoxin recovery from OVDs poses particular challenge due to the nature and composition of the device. In the present proof-of-concept study, we demonstrate real-time detection capability of endotoxin by employing a noncontact and label-free fiber-optic Fourier transform infrared transmission spectroscopy based sensing method in the midinfrared spectral range of 1.6–12 μm. We performed testing on OVD samples spiked with a series of different concentrations of endotoxin. The study suggested that endotoxin contamination in OVD might be associated with fingerprint spectral peak shift in the wavenumber ranges of $\text{2925}- \text{2890}\,\text{cm}^{- 1}$ and $\text{1125}-\text{1100}\,\text{cm}^{- 1}$ , and the distance of shifting is dependent upon endotoxin concentration in OVD. FO-FTIR integrated with multivariate approaches such as hierarchical clustering and principal component analysis provides significant differentiation of OVD, endotoxin, and contaminated OVD at different concentrations.

Bacterial contamination on medical device surfaces is a critical public health concern. In order to detect bacteria on medical device surface, alternative methods for quantitative, accurate, easy-to-use, and real-time detection and identification of microorganism contamination are needed. We have recently presented a novel proof-of-concept platform for non-contact, label-free and real-time detection of surface contamination employing a fiber-optic Fourier transform infrared (FO-FTIR) spectroscopy sensing methodology in the mid-infrared (mid-IR) spectral range of 1.6–12 μm. In the present study, we demonstrate the detection capability and sensitivity of the integrated FO-FTIR approach using four species of commonly encountered bacteria: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae. FO-FTIR combined with multivariate approaches such as hierarchical clustering and principal component analysis provided specific mid-IR spectral differentiation of the four microorganisms including when the sample contained mixtures of bacteria types. To assess the sensitivity of the FO-FTIR platform, bacteria samples were prepared at 109 colony forming unit (CFU)/μL and then serially diluted 1:10 eight times. The salient findings of this investigation showed that the integrated FO-FTIR based sensor can detect the presence of the bacteria at concentrations between 103 and 104 CFU/2 μL, producing unique bacteria signatures with high reproducibility. The advanced features of this sensing method in terms of sensitivity, specificity and repeatability employing non-contact, label-free, and real-time approaches, demonstrate its potential use as an alternative effective screening tool for routine monitoring of bacterial contaminated surfaces.

An advanced sensing methodology based on a noninvasive label-free fingerprint infrared spectroscopy approach for detecting and identifying intrinsic biomarkers related to the safety and efficacy of optical diagnostics and therapeutics technologies and devices is presented.

Ophthalmic Viscosurgical Devices (OVDs) in clinical setting are a major health risk factor for potential endotoxin contamination in the eye, due to their extensive applications in cataract surgery for space creation, stabilization and protection of intraocular tissue and intraocular lens (IOL) during implantation. Endotoxin contamination of OVDs is implicated in toxic anterior syndrome (TASS), a severe complication of cataract surgery that leads to intraocular damage and even blindness. Current standard methods for endotoxin contamination detection utilize rabbit assay or Limulus amoebocyte lysate (LAL) assays. These endotoxin detection strategies are extremely difficult for gel-like type devices such as OVDs. To overcome the endotoxin detection limitations in OVDs, we have developed an alternative optical detection methodology for label-free and real-time sensing of bacterial endotoxin in OVDs, based on fiber-optic Fourier transform infrared (FO-FTIR) transmission spectrometry in the mid-IR spectral range from 2.5 micron to 12 micron. Endotoxin contaminated OVD test samples were prepared by serial dilutions of endotoxins on OVDs. The major results of this study revealed two salient spectral peak shifts (in the regions 2925 to 2890 cm^-1 and 1125 to 1100 cm^-1), which are associated with endotoxin in OVDs. In addition, FO-FTIR experimental results processed using a multivariate analysis confirmed the observed specific peak shifts associated with endotoxin contamination in OVDs. Thus, employing the FO-FTIR sensing methodology integrated with a multivariate analysis could potentially be used as an alternative endotoxin detection technique in OVD.

Biophotonics is an emerging field in modern biomedical technology that has opened up new horizons for transfer of state-of-the-art techniques from the areas of lasers, fiber optics and biomedical optics to the life sciences and medicine. This field continues to vastly expand with advanced developments across the entire spectrum of biomedical applications ranging from fundamental “bench” laboratory studies to clinical patient “bedside” diagnostics and therapeutics. However, in order to translate these technologies to clinical device applications, the scientific and industrial community, and FDA are facing the requirement for a thorough evaluation and review of laser radiation safety and efficacy concerns. In many cases, however, the review process is complicated due the lack of effective means and standard test methods to precisely analyze safety and effectiveness of some of the newly developed biophotonics techniques and devices. There is, therefore, an immediate public health need for new test protocols, guidance documents and standard test methods to precisely evaluate fundamental characteristics, performance quality and safety of these technologies and devices. Here, we will overview our recent developments of novel test methodologies for safety and efficacy evaluation of some emerging biophotonics technologies and medical devices. These methodologies are based on integrating the advanced features of state-of-the-art optical sensor technologies and approaches such as high-resolution fiber-optic sensing, confocal and optical coherence tomography imaging, and infrared spectroscopy. The presentation will also illustrate some methodologies developed and implemented for testing intraocular lens implants, biochemical contaminations of medical devices, ultrahigh-resolution nanoscopy, and femtosecond laser therapeutics.

The major goal in developing drugs targeting specific tumor receptors, such as Monoclonal AntiBodies (MAB), is to make a drug compound that targets selectively the cancer-causing biomarkers, inhibits their functionality, and/or delivers the toxin specifically to the malignant cells. Recent advances in MABs show that their efficacy depends strongly on characterization of tumor biomarkers. Therefore, one of the main tasks in cancer diagnostics and treatment is to develop non-invasive in-vivo imaging techniques for detection of cancer biomarkers and monitoring their down regulation during the treatment. Such methods can potentially result in a new imaging and treatment paradigm for cancer therapy. In this article we have reviewed fluorescence imaging approaches, including those developed in our group, to detect and monitor Human Epidermal Growth Factor 2 (HER2) receptors before and during therapy. Transition of these techniques from the bench to bedside is the ultimate goal of our project. Similar approaches can be used potentially for characterization of other cancer related cell biomarkers.

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 (T1/2 < 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. Clin Cancer Res; 17(15); 5071–81. ©2011 AACR.

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.

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.

Introduction: Cancers overexpressing HER2 gene are usually described as more aggressive and are associated with poor prognosis. Although introduction of trastuzumab, a HER2-specific humanized antibody, significantly improved the outcome, there are still number of patients who do not respond or acquire resistance to HER2-targeted therapy. We have previously described the construction, expression, and in vitro characterization of Affitoxin, a recombinant protein combining HER2- specific Affibody and modified Pseudomonas exotoxin A (PE 38) that blocks protein synthesis by ADP ribosylation of eEF-2 (Zielinski et al. 2009). In this report we present in vivo data. Materials and Methods. Recombinant Affitoxins were expressed and purified from the soluble fraction of E.coli. HER2 specificity was confirmed in vitro by measurement of the residual ATP level in N87 cells treated with Affitoxin and “Non-specific” toxin. containing “off-target” instead of HER2 - Affibodies. Acute toxicity and immunogenicity studies were performed in female Balb/c mice. Affitoxin clearance rate was determined by ELISA and residual serum toxicity. Biodistribution in subcutaneous BT474 tumors model was determined by NIR optical imaging. In vivo efficacy of the drug was verified in subcutaneous (BT474, SKOV3) and intraperitoneal (SKOV3-luc-D3) tumor models. Results. In this work we have confirmed that HER2-specific Affitoxin is a potent agent eliminating HER2-overexpressing cells in vitro at low picomolar concentration range, while non-specific toxin-containing offtarget Affitoxin is four orders of magnitude less efficient. Acute toxicity studies showed that Affitoxin is well tolerated by mice with LD50 = 0.572±0.051 mg/kg. Injection of six doses every third day neither lead to significant weigh loss of the animal nor induced severe liver problems. Pharmacokinetic data indicated fast clearance of Affitoxin from the circulation with half-life shorter than 10 min. Using human tumor xenograft models, we have shown that Affitoxin accumulates in HER2- overexpressing BT474 tumors, leading not only to reduction of growth rate but also permanent shrinkage of relatively large ( Immunogenicity studies indicated that Affitoxin is less immunogenic than toxin containing antibody fragments fused to PE38 (HA22). Conclusions. Affitoxin is a novel recombinant protein, using high affinity Affibody molecules to target HER2 receptors. Our in vitro and in vivo data strongly suggest that Affitoxin might be an attractive alternative or complement for existing HER2 targeted therapies Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-23.

In this paper we examine possible data types for time resolved fluorescence enhanced diffuse optical tomography (FDOT). FDOT is a particular case of diffuse optical tomography, where our goal is to analyze fluorophores deeply embedded in a turbid medium. We focus on the relative robustness of the different sets of data types to noise. We use an analytical model to generate the expected temporal point spread function (TPSF) and generate the data types from this. Varying levels of noise are applied to the TPSF before generating the data types. We show that local data types are more robust to noise than global data types, and should provide enhanced information to the inverse problem. We go on to show that with a simple reconstruction algorithm, depth and lifetime (the parameters of interest) of the fluorophore are better reconstructed using the local data types. Further we show that the relationship between depth and lifetime is better preserved for the local data types, suggesting they are in some way not only more robust, but also self-regularizing. We conclude that while the local data types may be more expensive to generate in the general case, they do offer clear advantages over the standard global data types.

We demonstrate a novel integrated fiber-optic Fourier Transform Infrared (FO-FTIR) spectroscopy sensing approach for non-contact, rapid and chemical-free detection of pathogenic contamination on medical device surfaces. A proof-of-concept experimental sensing platform is validated through multivariable analytical studies.

Here we present detailed transmission measurements of ex-vivo lens capsule from 40,000 cm−1 to 800 cm−1. Time-dependent measurements were also performed to eliminate water absorption.

Two noninvasive methods, thermography and laser Doppler imaging (LDI), were assessed for their ability to quantitatively assess parameters of vascularity in lesions of HIV-associated Kaposi's sarcoma (KS). Thermography and LDI images of a representative KS lesion were recorded in 16 patients and compared to normal skin either adjacent to the lesion or on the contralateral side. Eleven of the 16 patients had greater than 0.5 °C increased temperature and 12 of the 16 patients had increased flux (measured by LDI) as compared to normal skin. There was a strong correlation between these two parameters (R = 0.81, p < 0.001). In ten patients, measurements were obtained prior to therapy and after receiving a regimen of liposomal doxorubicin and interleukin-12. After 18 weeks of therapy, temperature and blood flow of the lesions were significantly reduced from the baseline (p = 0.004 and 0.002 respectively). These techniques hold promise to assess physiologic parameters in KS lesions and their changes with therapy.

Optical methods have a long history in the field of medical diagnosis. The biomolecular specificity possible with optical methods has been particularly valuable in microscopy and histopathology while in vivo imaging of deep structures has traditionally been the domain of X-ray and MRI. The use of optical methods in deep tissue has been limited by multiple-scattering which blurs or distorts the optical signal. New stochastic methods which account for multiple scattering have been developed that are extending the usefulness of optical methods deep into tissue. In optical mammography, photons may travel through 10cm of tissue before arriving at the detector. We have developed a method for quantifying parameters of anomalous sites in breast tissue that may be used for functional characterization of tumors. In other work presented here, we are developing fluorescence based methods to detect and monitor tumor status. The immune response to a tumor is a target for fluorescently labeled specific antibodies. We have developed a method to localize the tumor site using CW fluorescence. Additionally, we have developed a method which uses time-resolved data and capitalizes on probe lifetime sensitivity to metabolic parameters such as pH and temperature to obtain functional information from the tumor site.

Our latest system for the imaging of temperature and other thermal properties of the skin provides not only precise temperature distribution of the skin but also high-speed measurement of emissivity and inertia distribution. The method enabled us to measure the emissivity and thermal inertia of the skin even when the temperature distribution of the inner surface of the hood was not uniform. Two-dimensional measurement with hood switching, and a single-hood measurement system were considered.

In vivo microwave treatments for bone tumor are designed, which enable us to conserve the activity and functionality of the matrix of living tissues. This treatment is composed of two steps. In the first step, the tumor was coagulated by the application of microwaves emitted from the antenna inserted into the tumor tissue, and then retrieved. In the second step, the surrounding tissue suspected to be invaded with transformed cells was covered with hydro gels and heated similarly. The tissue itself was heated by the conduction from the gels. The tissue temperature should be kept at 60°C for 30 minutes. This treatment should kill the whole cells within the tissues, but the mechanical strength and the biochemical activity of the matrix should be left intact. The matrix preserves the mechanical functions and ensures the maximum regeneration ability of the tissue. In this study, various hydro gels were examined and the most promising one was selected. Animal experiments were carried out and successful heating verified the applicability of the 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. Clin Cancer Res; 20(13); 3531–9. ©2014 AACR.

In this study we have shown that in-vivo fluorescence lifetime imaging can be used for monitoring the efficacy of targeted therapy with 17-DMAG on HER2 positive tumors.

Pharmacokinetic analysis of images, obtained after injection of Her2-specific fluorescent probes, relates the probe accumulation rate in tumors to Her2 overexpression. Correlation between fluorescence lifetime and probe binding to Her2 is discussed.

We present a novel approach for non-contact, rapid and chemical-free decontamination of medical device surfaces using near-infrared ultrashort (nano- to femtosecond) pulse lasers. A proof-of-principal experimental platform is validated through multivariable comparison studies.

An attempt was made to image the thermal inertia (defined as the square root of the product of thermal conductivity, specific heat and density) of the skin to observe the distribution of blood in the skin during post-occlusive reactive hyperaemia in normal healthy volunteers. The method was based on the ability to calculate thermal inertia by successive thermographic measurements of the skin after stepwise change in ambient radiation temperature surrounding the skin area. The stepwise change was achieved within 0.1 s through a single hood. Experimentation on the undisturbed volar forearm of normal subjects at the same site showed that the measurements thus achieved were reproducible. The thermal inertia values of forearm skin in normal subjects were scattered throughout the range 1.1 x 10(3) to 1.7 x 10(3) W s(1/2) m(-2) K(-1). Experiments on forearm skin subjected to arterial cuff occlusion indicated that thermal inertia can be detected at a low level of blood perfusion. A linear relationship was observed between thermal inertia and blood perfusion measured by laser Doppler imager before and during blood flow occlusion. During reactive hyperaemia, the thermal inertia image exhibited a non-uniform island-shaped pattern of distribution over the forearm, suggesting that, after release from occlusion, recovery of blood flow is non-uniform.

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.

Electrical impedance tomography (EIT) is a tubeless technique that generates tomographic images of gastric resistivity. We investigated the application of EIT to measure gastric acid secretion. Nineteen normal subjects underwent a standard intubation test. Basal acid output (BAO) and stimulated acid output (SAO) (millimoles per hour) were measured before and after pentagastrin, respectively. On a different day, EIT was performed before (basal) and after pentagastrin (stimulated). The changes in impedance over time were measured and the area under the curve (AUC) was calculated. Both the tests were repeated in 13 subjects after omeprazole treatment. As in the intubation test, there was the expected increase in AUC value after pentagastrin (basal vs stimulated; 1.2 +/- 2.8 vs 731 +/- 297, P < 0.0001). A significant fall in acid output and AUC following omeprazole pretreatment was observed (without vs with omeprazole; 20.5 +/- 5.7 vs 0.03 +/- 0.06, P < 0.0001 for intubation test and 731 +/- 297 vs 44 +/- 172, P < 0.0001 for EIT). There was a significant correlation between SAO and the delta AUC with (r = 0.65 P < 0.001) or without (r = 0.95, P < 0.001) omeprazole and in all the experiments (r = 0.87, P < 0.001). This study demonstrates the predictable change of gastric impedance and may be useful as a noninvasive test for measuring gastric acid secretion.

Electrical impedance tomography (EIT) is designed essentially for two-dimensional imaging, but current flow in the third dimension causes images to be formed for objects in 3D. The present work has shown that the image of an object is shifted in position towards the centre almost linearly with the 3D distance from the electrode plane and that the slope of this linear variation depends on the radial distance of the object. An empirical curve has been fitted to this dependence, based on which a method has been developed to locate 3D point objects from EIT measurements in only two planes. This will be useful in clinical and other applications in which 3D objects are few and widely separated. This new methodology may be the basis for 3D imaging in the future.

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.

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.

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.

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 (KD = 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.

The designed polarization adapter is incorporated into a colposcope provides illumination with polarized light and capturing of orthogonally polarized images, using liquid crystal retarder and polarizer. Pearson correlation analysis is used to characterize subsurface structures.

Assessment of the response of Kaposi’s sarcoma lesions to therapy is challenging. We propose diffuse multi-spectral imaging for lesion follow-up as an objective measure for treatment efficacy.