Publications by authors named "Nimmi Ramanujam"

43 Publications

Optimizing ethyl cellulose-ethanol delivery towards enabling ablation of cervical dysplasia.

Sci Rep 2021 08 19;11(1):16869. Epub 2021 Aug 19.

Department of Biomedical Engineering, Duke University, Durham, NC, USA.

In low-income countries, up to 80% of women diagnosed with cervical dysplasia do not return for follow-up care, primarily due to treatment being inaccessible. Here, we describe development of a low-cost, portable treatment suitable for such settings. It is based on injection of ethyl cellulose (EC)-ethanol to ablate the transformation zone around the os, the site most impacted by dysplasia. EC is a polymer that sequesters the ethanol within a prescribed volume when injected into tissue, and this is modulated by the injected volume and delivery parameters (needle gauge, bevel orientation, insertion rate, depth, and infusion rate). Salient injection-based delivery parameters were varied in excised swine cervices. The resulting injection distribution volume was imaged with a wide-field fluorescence imaging device or computed tomography. A 27G needle and insertion rate of 10 mm/s achieved the desired insertion depth in tissue. Orienting the needle bevel towards the outer edge of the cervix and keeping infusion volumes ≤ 500 µL minimized leakage into off-target tissue. These results guided development of a custom hand-held injector, which was used to locate and ablate the upper quadrant of a swine cervix in vivo with no adverse events or changes in host temperature or heart rate. After 24 h, a distinct region of necrosis was detected that covered a majority (> 75%) of the upper quadrant of the cervix, indicating four injections could effectively cover the full cervix. The work here informs follow up large animal in vivo studies, e.g. in swine, to further assess safety and efficacy of EC-ethanol ablation in the cervix.
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http://dx.doi.org/10.1038/s41598-021-96223-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376953PMC
August 2021

KeyLoop: Mechanical Retraction of the Abdominal Wall for Gasless Laparoscopy.

Surg Innov 2021 Jul 9:15533506211031084. Epub 2021 Jul 9.

Duke Global Health Institute, Durham, NC, USA.

Despite favorable outcomes of laparoscopic surgery in high-income countries, its implementation in low- and middle-income countries (LMICs) is challenging given a shortage of consumable supplies, high cost, and risk of power outages. To overcome these barriers, we designed a mechanical retractor that provides vertical tension on the anterior abdominal wall. The retractor design is anatomically and mathematically optimized to provide exposure similar to traditional gas-based insufflation methods. Anatomical data from computed tomography scans were used to define retractor size. The retractor is constructed of biocompatible stainless steel rods and paired with a table-mounted lifting system to provide 5 degrees of freedom. Structural integrity was assessed through finite element analysis (FEA) and load testing. Functional testing was performed in a laparotomy model. A user guide based on patient height and weight was created to customize retractor size, and 4 retractor sizes were constructed. FEA data using a 13.6 kg mass (15 mm Hg pneumoperitoneum) show a maximum of 30 mm displacement with no permanent deformation. Physical load testing with applied weight from 0 to 13.6 kg shows a maximum of 60 mm displacement, again without permanent deformation. Retraction achieved a 57% larger field of view compared to an unretracted state in a laparotomy model. The KeyLoop retractor maintains structural integrity, is easily sterilized, and can be readily manufactured, making it a viable alternative to traditional insufflation methods. For surgeons and patients in LMICs, the KeyLoop provides a means to increase access to laparoscopic surgery.
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http://dx.doi.org/10.1177/15533506211031084DOI Listing
July 2021

Digital Health Strategies for Cervical Cancer Control in Low- and Middle-Income Countries: Systematic Review of Current Implementations and Gaps in Research.

J Med Internet Res 2021 05 27;23(5):e23350. Epub 2021 May 27.

Duke Global Health Institute, Durham, NC, United States.

Background: Nearly 90% of deaths due to cervical cancer occur in low- and middle-income countries (LMICs). In recent years, many digital health strategies have been implemented in LMICs to ameliorate patient-, provider-, and health system-level challenges in cervical cancer control. However, there are limited efforts to systematically review the effectiveness and current landscape of digital health strategies for cervical cancer control in LMICs.

Objective: We aim to conduct a systematic review of digital health strategies for cervical cancer control in LMICs to assess their effectiveness, describe the range of strategies used, and summarize challenges in their implementation.

Methods: A systematic search was conducted to identify publications describing digital health strategies for cervical cancer control in LMICs from 5 academic databases and Google Scholar. The review excluded digital strategies associated with improving vaccination coverage against human papillomavirus. Titles and abstracts were screened, and full texts were reviewed for eligibility. A structured data extraction template was used to summarize the information from the included studies. The risk of bias and data reporting guidelines for mobile health were assessed for each study. A meta-analysis of effectiveness was planned along with a narrative review of digital health strategies, implementation challenges, and opportunities for future research.

Results: In the 27 included studies, interventions for cervical cancer control focused on secondary prevention (ie, screening and treatment of precancerous lesions) and digital health strategies to facilitate patient education, digital cervicography, health worker training, and data quality. Most of the included studies were conducted in sub-Saharan Africa, with fewer studies in other LMIC settings in Asia or South America. A low risk of bias was found in 2 studies, and a moderate risk of bias was found in 4 studies, while the remaining 21 studies had a high risk of bias. A meta-analysis of effectiveness was not conducted because of insufficient studies with robust study designs and matched outcomes or interventions.

Conclusions: Current evidence on the effectiveness of digital health strategies for cervical cancer control is limited and, in most cases, is associated with a high risk of bias. Further studies are recommended to expand the investigation of digital health strategies for cervical cancer using robust study designs, explore other LMIC settings with a high burden of cervical cancer (eg, South America), and test a greater diversity of digital strategies.
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http://dx.doi.org/10.2196/23350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8193495PMC
May 2021

An Accessible Laparoscope for Surgery in Low- and Middle- Income Countries.

Ann Biomed Eng 2021 Jul 8;49(7):1657-1669. Epub 2021 Mar 8.

Duke Global Health Institute, Durham, NC, USA.

Laparoscopic surgery is the standard of care in high-income countries for many procedures in the chest and abdomen. It avoids large incisions by using a tiny camera and fine instruments manipulated through keyhole incisions, but it is generally unavailable in low- and middle-income countries (LMICs) due to the high cost of installment, lack of qualified maintenance personnel, unreliable electricity, and shortage of consumable items. Patients in LMICs would benefit from laparoscopic surgery, as advantages include decreased pain, improved recovery time, fewer wound infections, and shorter hospital stays. To address this need, we developed an accessible laparoscopic system, called the ReadyView laparoscope for use in LMICs. The device includes an integrated camera and LED light source that can be displayed on any monitor. The ReadyView laparoscope was evaluated with standard optical imaging targets to determine its performance against a state-of-the-art commercial laparoscope. The ReadyView laparoscope has a comparable resolving power, lens distortion, field of view, depth of field, and color reproduction accuracy to a commercially available endoscope, particularly at shorter, commonly-used working distances (3-5 cm). Additionally, the ReadyView has a cooler temperature profile, decreasing the risk for tissue injury and operating room fires. The ReadyView features a waterproof design, enabling sterilization by submersion, as commonly performed in LMICs. A custom desktop software was developed to view the video on a laptop computer with a frame rate greater than 30 frames per second and to white balance the image, which is critical for clinical use. The ReadyView laparoscope is capable of providing the image quality and overall performance needed for laparoscopic surgery. This portable low-cost system is well suited to increase access to laparoscopic surgery in LMICs.
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http://dx.doi.org/10.1007/s10439-020-02707-6DOI Listing
July 2021

Understanding Factors Governing Distribution Volume of Ethyl Cellulose-Ethanol to Optimize Ablative Therapy in the Liver.

IEEE Trans Biomed Eng 2020 08 16;67(8):2337-2348. Epub 2019 Dec 16.

Objective: Ethanol ablation, the injection of ethanol to induce necrosis, was originally used to treat hepatocellular carcinoma, with survival rates comparable to surgery. However, efficacy is limited due to leakage into surrounding tissue. To reduce leakage, we previously reported incorporating ethyl cellulose (EC) with ethanol as this mixture forms a gel when injected into tissue. To further develop EC-ethanol injection as an ablative therapy, the present study evaluates the extent to which salient injection parameters govern the injected fluid distribution.

Methods: Utilizing ex vivo swine liver, injection parameters (infusion rate, EC%, infusion volume) were examined with fluorescein added to each solution. After injection, tissue samples were frozen, sectioned, and imaged.

Results: While leakage was higher for ethanol and 3%EC-ethanol at a rate of 10 mL/hr compared to 1 mL/hr, leakage remained low for 6%EC-ethanol regardless of infusion rate. The impact of infusion volume and pressure were also investigated first in tissue-mimicking surrogates and then in tissue. Results indicated that there is a critical infusion pressure beyond which crack formation occurs leading to fluid leakage. At a rate of 10 mL/hr, a volume of 50 μL remained below the critical pressure.

Conclusions: Although increasing the infusion rate increases stress on the tissue and the risk of crack formation, injections of 6%EC-ethanol were localized regardless of infusion rate. To further limit leakage, multiple low-volume infusions may be employed.

Significance: These results, and the experimental framework developed to obtain them, can inform optimizing EC-ethanol to treat a range of medical conditions.
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http://dx.doi.org/10.1109/TBME.2019.2960049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295656PMC
August 2020

Development of Algorithms for Automated Detection of Cervical Pre-Cancers With a Low-Cost, Point-of-Care, Pocket Colposcope.

IEEE Trans Biomed Eng 2019 08 18;66(8):2306-2318. Epub 2018 Dec 18.

Goal: In this paper, we propose methods for (1) automatic feature extraction and classification for acetic acid and Lugol's iodine cervigrams and (2) methods for combining features/diagnosis of different contrasts in cervigrams for improved performance.

Methods: We developed algorithms to pre-process pathology-labeled cervigrams and extract simple but powerful color and textural-based features. The features were used to train a support vector machine model to classify cervigrams based on corresponding pathology for visual inspection with acetic acid, visual inspection with Lugol's iodine, and a combination of the two contrasts.

Results: The proposed framework achieved a sensitivity, specificity, and accuracy of 81.3%, 78.6%, and 80.0%, respectively, when used to distinguish cervical intraepithelial neoplasia (CIN+) relative to normal and benign tissues. This is superior to the average values achieved by three expert physicians on the same data set for discriminating normal/benign cases from CIN+ (77% sensitivity, 51% specificity, and 63% accuracy).

Conclusion: The results suggest that utilizing simple color- and textural-based features from visual inspection with acetic acid and visual inspection with Lugol's iodine images may provide unbiased automation of cervigrams.

Significance: This would enable automated, expert-level diagnosis of cervical pre-cancer at the point of care.
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http://dx.doi.org/10.1109/TBME.2018.2887208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581620PMC
August 2019

Simultaneous in vivo optical quantification of key metabolic and vascular endpoints reveals tumor metabolic diversity in murine breast tumor models.

J Biophotonics 2019 04 28;12(4):e201800372. Epub 2019 Jan 28.

Department of Biomedical Engineering, Duke University, Durham, North Carolina.

Therapeutically exploiting vascular and metabolic endpoints becomes critical to translational cancer studies because altered vascularity and deregulated metabolism are two important cancer hallmarks. The metabolic and vascular phenotypes of three sibling breast tumor lines with different metastatic potential are investigated in vivo with a newly developed quantitative spectroscopy system. All tumor lines have different metabolic and vascular characteristics compared to normal tissues, and there are strong positive correlations between metabolic (glucose uptake and mitochondrial membrane potential) and vascular (oxygen saturations and hemoglobin concentrations) parameters for metastatic (4T1) tumors but not for micrometastatic (4T07) and nonmetastatic (67NR) tumors. A longitudinal study shows that both vascular and metabolic endpoints of 4T1 tumors increased up to a specific tumor size threshold beyond which these parameters decreased. The synchronous changes between metabolic and vascular parameters, along with the strong positive correlations between these endpoints suggest that 4T1 tumors rely on strong oxidative phosphorylation in addition to glycolysis. This study illustrates the great potential of our optical technique to provide valuable dynamic information about the interplay between the metabolic and vascular status of tumors, with important implications for translational cancer investigations.
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http://dx.doi.org/10.1002/jbio.201800372DOI Listing
April 2019

Near-simultaneous quantification of glucose uptake, mitochondrial membrane potential, and vascular parameters in murine flank tumors using quantitative diffuse reflectance and fluorescence spectroscopy.

Biomed Opt Express 2018 Jul 27;9(7):3399-3412. Epub 2018 Jun 27.

Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

The shifting metabolic landscape of aggressive tumors, with fluctuating oxygenation conditions and temporal changes in glycolysis and mitochondrial metabolism, is a critical phenomenon to study in order to understand negative treatment outcomes. Recently, we have demonstrated near-simultaneous optical imaging of mitochondrial membrane potential (MMP) and glucose uptake in non-tumor window chambers, using the fluorescent probes tetramethylrhodamine ethyl ester (TMRE) and 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Here, we demonstrate a complementary technique to perform near-simultaneous optical spectroscopy of tissue vascular parameters, glucose uptake, and MMP in a solid tumor model that is most often used for therapeutic studies. Our study demonstrates the potential of optical spectroscopy as an effective tool to quantify the vascular and metabolic characteristics of a tumor, which is an important step towards understanding the mechanisms underlying cancer progression, metastasis, and resistance to therapies.
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http://dx.doi.org/10.1364/BOE.9.003399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033552PMC
July 2018

An integrated strategy for improving contrast, durability, and portability of a Pocket Colposcope for cervical cancer screening and diagnosis.

PLoS One 2018 9;13(2):e0192530. Epub 2018 Feb 9.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

Introduction: We have previously developed a portable Pocket Colposcope for cervical cancer screening in resource-limited settings. In this manuscript we report two different strategies (cross-polarization and an integrated reflector) to improve image contrast levels achieved with the Pocket Colposcope and evaluate the merits of each strategy compared to a standard-of-care digital colposcope. The desired outcomes included reduced specular reflection (glare), increased illumination beam pattern uniformity, and reduced electrical power budget. In addition, anti-fogging and waterproofing features were incorporated to prevent the Pocket Colposcope from fogging in the vaginal canal and to enable rapid disinfection by submersion in chemical agents.

Methods: Cross-polarization (Generation 3 Pocket Colposcope) and a new reflector design (Generation 4 Pocket Colposcope) were used to reduce glare and improve contrast. The reflector design (including the angle and height of the reflector sidewalls) was optimized through ray-tracing simulations. Both systems were characterized with a series of bench tests to assess specular reflection, beam pattern uniformity, and image contrast. A pilot clinical study was conducted to compare the Generation 3 and 4 Pocket Colposcopes to a standard-of-care colposcope (Leisegang Optik 2). Specifically, paired images of cervices were collected from the standard-of-care colposcope and either the Generation 3 (n = 24 patients) or the Generation 4 (n = 32 patients) Pocket Colposcopes. The paired images were blinded by device, randomized, and sent to an expert physician who provided a diagnosis for each image. Corresponding pathology was obtained for all image pairs. The primary outcome measures were the level of agreement (%) and κ (kappa) statistic between the standard-of-care colposcope and each Pocket Colposcope (Generation 3 and Generation 4).

Results: Both generations of Pocket Colposcope had significantly higher image contrast when compared to the standard-of-care colposcope. The addition of anti-fog and waterproofing features to the Generation 3 and 4 Pocket Colposcope did not impact image quality based on qualitative and quantitative metrics. The level of agreement between the Generation 3 Pocket Colposcope and the standard-of-care colposcope was 75.0% (kappa = 0.4000, p = 0.0028, n = 24). This closely matched the level of agreement between the Generation 4 Pocket Colposcope and the standard-of-care colposcope which was also 75.0% (kappa = 0.4941, p = 0.0024, n = 32).

Conclusion: Our results indicate that the Generation 3 and 4 Pocket Colposcopes perform comparably to the standard-of-care colposcope, with the added benefit of being low-cost and waterproof, which is ideal for use in resource-limited settings. Additionally, the reflector significantly reduces the electrical requirements of the Generation 4 Pocket Colposcope enhancing portability without altering performance compared to the Generation 3 system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192530PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806883PMC
April 2018

Near-simultaneous intravital microscopy of glucose uptake and mitochondrial membrane potential, key endpoints that reflect major metabolic axes in cancer.

Sci Rep 2017 10 23;7(1):13772. Epub 2017 Oct 23.

Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA.

While the demand for metabolic imaging has increased in recent years, simultaneous in vivo measurement of multiple metabolic endpoints remains challenging. Here we report on a novel technique that provides in vivo high-resolution simultaneous imaging of glucose uptake and mitochondrial metabolism within a dynamic tissue microenvironment. Two indicators were leveraged; 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) reports on glucose uptake and Tetramethylrhodamine ethyl ester (TMRE) reports on mitochondrial membrane potential. Although we demonstrated that there was neither optical nor chemical crosstalk between 2-NBDG and TMRE, TMRE uptake was significantly inhibited by simultaneous injection with 2-NBDG in vivo. A staggered delivery scheme of the two agents (TMRE injection was followed by 2-NBDG injection after a 10-minute delay) permitted near-simultaneous in vivo microscopy of 2-NBDG and TMRE at the same tissue site by mitigating the interference of 2-NBDG with normal glucose usage. The staggered delivery strategy was evaluated under both normoxic and hypoxic conditions in normal tissues as well as in a murine breast cancer model. The results were consistent with those expected for independent imaging of 2-NBDG and TMRE. This optical imaging technique allows for monitoring of key metabolic endpoints with the unique benefit of repeated, non-destructive imaging within an intact microenvironment.
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http://dx.doi.org/10.1038/s41598-017-14226-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653871PMC
October 2017

Distinct Angiogenic Changes during Carcinogenesis Defined by Novel Label-Free Dark-Field Imaging in a Hamster Cheek Pouch Model.

Cancer Res 2017 12 11;77(24):7109-7119. Epub 2017 Oct 11.

Department of Biomedical Engineering, Duke University, Durham, North Carolina.

There remain gaps in knowledge concerning how vascular morphology evolves during carcinogenesis. In this study, we imaged neovascularization by label-free dark-field microscopy of a 7,12-Dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch model of oral squamous cell carcinoma (SCC). Wavelength-dependent imaging revealed distinct vascular features at different imaging depths and vessel sizes. Vascular tortuosity increased significantly in high-risk lesions, whereas diameter decreased significantly in hyperplastic and SCC lesions. Large vessels preserved the same trends seen in the original images, whereas small vessels displayed different trends, with length and diameter increasing during carcinogenesis. On the basis of these data, we developed and validated a classification algorithm incorporating vascular features from different vessel masks. Receiver operator curves generated from the classification results demonstrated high accuracies in discriminating normal and hyperplasia from high-grade lesions (AUC > 0.94). Overall, these results provided automated imaging of vasculature in the earliest stages of carcinogenesis from which one can extract robust endpoints. The optical toolbox described here is simple, low-cost and portable, and can be used in a variety of health care and research settings for cancer prevention and pharmacology research. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221460PMC
December 2017

Design and preliminary analysis of a vaginal inserter for speculum-free cervical cancer screening.

PLoS One 2017 31;12(5):e0177782. Epub 2017 May 31.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

Objective: Cervical cancer screening usually requires use of a speculum to provide a clear view of the cervix. The speculum is one potential barrier to screening due to fear of pain, discomfort and embarrassment. The aim of this paper is to present and demonstrate the feasibility of a tampon-sized inserter and the POCkeT Colposcope, a miniature pen sized-colposcope, for comfortable, speculum-free and potentially self-colposcopy.

Study Design: We explored different designs using 3D computer-aided design (CAD) software and performed mechanical testing simulations on each. Designs were rapid prototyped and tested using a custom vaginal phantom across a range of vaginal pressures and uterine tilts to select an optimal design. Two final designs were tested with fifteen volunteers to assess cervix visualization, comfort and usability compared to the speculum and the optimal design, the curved-tip inserter, was selected for testing in volunteers.

Results: We present a vaginal inserter as an alternative to the standard speculum for use with the POCkeT Colposcope. The device has a slim tubular body with a funnel-like curved tip measuring approximately 2.5 cm in diameter. The inserter has a channel through which a 2 megapixel (MP) mini camera with LED illumination fits to enable image capture. Mechanical finite element testing simulations with an applied pressure of 15 cm H2O indicated a high factor of safety (90.9) for the inserter. Testing of the device with a custom vaginal phantom, across a range of supine vaginal pressures and uterine tilts (retroverted, anteverted and sideverted), demonstrated image capture with a visual area comparable to the speculum for a normal/axial positioned uteri and significantly better than the speculum for anteverted and sideverted uteri (p<0.00001). Volunteer studies with self-insertion and physician-assisted cervix image capture showed adequate cervix visualization for 83% of patients. In addition, questionnaire responses from volunteers indicated a 92.3% overall preference for the inserter over the speculum and all indicated that the inserter was more comfortable than the speculum. The inserter provides a platform for self-cervical cancer screening and also enables acetic acid/Lugol's iodine application and insertion of swabs for Pap smear sample collection.

Conclusion: This study demonstrates the feasibility of an inserter and miniature-imaging device for comfortable cervical image capture of women with potential for synergistic HPV and Pap smear sample collection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177782PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451045PMC
September 2017

International Image Concordance Study to Compare a Point-of-Care Tampon Colposcope With a Standard-of-Care Colposcope.

J Low Genit Tract Dis 2017 Apr;21(2):112-119

1Department of Biomedical Engineering, Duke University, Durham, NC; 2Duke Global Health Institute, Duke University, Durham, NC; 3Department of Surgery, Duke University Medical Center, Durham, NC; 4Department of Biostatistics and Bioinformatics, Duke University Medical School, Durham, NC; 5Institute of Cytology and Preventative Oncology (ICMR), New Delhi, India; 6Cancer Institute (WIA), Chennai, India; 7Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC; 8Kilimanjaro Christian Medical Centre, Moshi, Tanzania; 9Liga Contra el Cáncer, Pueblo Libre, Lima, Peru; and 10Department of Obstetrics and Gynecology, School of Medicine, Kenyatta University, Nairobi, Kenya.

Objective: Barriers to cervical cancer screening in low-resource settings include lack of accessible, high-quality services, high cost, and the need for multiple visits. To address these challenges, we developed a low-cost, intravaginal, optical cervical imaging device, the point-of-care tampon (POCkeT) colposcope and evaluated whether its performance is comparable with a standard-of-care colposcope.

Materials And Methods: There were 2 protocols, which included 44 and 18 patients. For the first protocol, white-light cervical images were collected in vivo, blinded by device, and sent electronically to 8 physicians from high-, middle-, and low-income countries. For the second protocol, green-light images were also collected and sent electronically to the highest performing physician from the first protocol who has experience in both a high- and low-income country. For each image, physicians completed a survey assessing cervix characteristics and severity of precancerous lesions. Corresponding pathology was obtained for all image pairs.

Results: For the first protocol, average percent agreement between devices was 70% across all physicians. The POCkeT and standard-of-care colposcope images had 37% and 51% agreement with pathology for high-grade squamous intraepithelial lesions (HSILs), respectively. Investigation of HSIL POCkeT images revealed decreased visibility of vascularization and lack of contrast in lesion margins. After changes were made for the second protocol, the 2 devices achieved similar agreement to pathology for HSIL lesions (55%).

Conclusions: Based on the exploratory study, physician interpretation of cervix images acquired using a portable, low-cost POCkeT colposcope was comparable to a standard-of-care colposcope.
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http://dx.doi.org/10.1097/LGT.0000000000000306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365351PMC
April 2017

Miniature spectral imaging device for wide-field quantitative functional imaging of the morphological landscape of breast tumor margins.

J Biomed Opt 2017 02;22(2):26007

Duke University, Pratt School of Engineering, Department of Biomedical Engineering, Durham, North Carolina, United States.

We have developed a portable, breast margin assessment probe leveraging diffuse optical spectroscopy to quantify the morphological landscape of breast tumor margins during breast conserving surgery. The approach presented here leverages a custom-made 16-channel annular photodiode imaging array (arranged in a 4 × 4 grid), a raster-scanning imaging platform with precision pressure control, and compressive sensing with an optimized set of eight wavelengths in the visible spectral range. A scalable Monte-Carlo-based inverse model is used to generate optical property [ ? s ? ( ? ) and ? a ( ? ) ] measures for each of the 16 simultaneously captured diffuse reflectance spectra. Subpixel sampling (0.75 mm) is achieved through incremental x , y raster scanning of the imaging probe, providing detailed optical parameter maps of breast margins over a 2 × 2 ?? cm 2 area in ? 9 ?? min . The morphological landscape of a tumor margin is characterized using optical surrogates for the fat to fibroglandular content ratio, which has demonstrated diagnostic utility in delineating tissue subtypes in the breast.
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http://dx.doi.org/10.1117/1.JBO.22.2.026007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321165PMC
February 2017

Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes.

Biomed Opt Express 2016 Sep 12;7(9):3412-3424. Epub 2016 Aug 12.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.

Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems.
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http://dx.doi.org/10.1364/BOE.7.003412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030020PMC
September 2016

Dark field optical imaging reveals vascular changes in an inducible hamster cheek pouch model during carcinogenesis.

Biomed Opt Express 2016 Sep 5;7(9):3247-3261. Epub 2016 Aug 5.

Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

In this study, we propose a low-cost cross-polarized dark field microscopy system for vascular imaging to detect head and neck cancer. A simple-to-use Gabor-filter-based image processing technique was developed to objectively and automatically quantify several important vascular features, including tortuosity, length, diameter and area fraction, from vascular images. Simulations were performed to evaluate the accuracies of vessel segmentation and feature extraction for our algorithm. Sensitivity and specificity for vessel segmentation of the Gabor masks both remained above 80% at all contrast levels when compared to gold-standard masks. Errors for vascular feature extraction were under 5%. Moreover, vascular contrast and vessel diameter were identified to be the two primary factors which affected the segmentation accuracies. After our algorithm was validated, we monitored the blood vessels in an inducible hamster cheek pouch carcinogen model over 17 weeks and quantified vascular features during carcinogenesis. A significant increase in vascular tortuosity and a significant decrease in vessel length were observed during carcinogenesis.
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http://dx.doi.org/10.1364/BOE.7.003247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030008PMC
September 2016

Oxygen and Perfusion Kinetics in Response to Fractionated Radiation Therapy in FaDu Head and Neck Cancer Xenografts Are Related to Treatment Outcome.

Int J Radiat Oncol Biol Phys 2016 10 14;96(2):462-469. Epub 2016 Jun 14.

: Department of Radiation Oncology, Duke University, Durham, NC, USA.

Purpose: To test whether oxygenation kinetics correlate with the likelihood for local tumor control after fractionated radiation therapy.

Methods And Materials: We used diffuse reflectance spectroscopy to noninvasively measure tumor vascular oxygenation and total hemoglobin concentration associated with radiation therapy of 5 daily fractions (7.5, 9, or 13.5 Gy/d) in FaDu xenografts. Spectroscopy measurements were obtained immediately before each daily radiation fraction and during the week after radiation therapy. Oxygen saturation and total hemoglobin concentration were computed using an inverse Monte Carlo model.

Results: First, oxygenation kinetics during and after radiation therapy, but before tumor volumes changed, were associated with local tumor control. Locally controlled tumors exhibited significantly faster increases in oxygenation after radiation therapy (days 12-15) compared with tumors that recurred locally. Second, within the group of tumors that recurred, faster increases in oxygenation during radiation therapy (day 3-5 interval) were correlated with earlier recurrence times. An area of 0.74 under the receiver operating characteristic curve was achieved when classifying the local control tumors from all irradiated tumors using the oxygen kinetics with a logistic regression model. Third, the rate of increase in oxygenation was radiation dose dependent. Radiation doses ≤9.5 Gy/d did not initiate an increase in oxygenation, whereas 13.5 Gy/d triggered significant increases in oxygenation during and after radiation therapy.

Conclusions: Additional confirmation is required in other tumor models, but these results suggest that monitoring tumor oxygenation kinetics could aid in the prediction of local tumor control after radiation therapy.
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http://dx.doi.org/10.1016/j.ijrobp.2016.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130289PMC
October 2016

Correlation of breast tissue histology and optical signatures to improve margin assessment techniques.

J Biomed Opt 2016 06;21(6):66014

Duke University Medical Center, Department of Pathology, DUMC3712, 200 Trent Drive, Durham, North Carolina 27710, United States.

Optical spectroscopy is sensitive to morphological composition and has potential applications in intraoperative margin assessment. Here, we evaluate ex vivo breast tissue and corresponding quantified hematoxylin & eosin images to correlate optical scattering signatures to tissue composition stratified by patient characteristics. Adipose sites (213) were characterized by their cell area and density. All other benign and malignant sites (181) were quantified using a grid method to determine composition. The relationships between mean reduced scattering coefficient (〈μs′〉), and % adipose, % collagen, % glands, adipocyte cell area, and adipocyte density were investigated. These relationships were further stratified by age, menopausal status, body mass index (BMI), and breast density. We identified a positive correlation between 〈μs′〉 and % collagen and a negative correlation between 〈μs′〉 and age and BMI. Increased collagen corresponded to increased 〈μs′〉 variability. In postmenopausal women, 〈μs′〉 was similar regardless of fibroglandular content. Contributions from collagen and glands to 〈μs′〉 were independent and equivalent in benign sites; glands showed a stronger positive correlation than collagen to 〈μs′〉 in malignant sites. Our data suggest that scattering could differentiate highly scattering malignant from benign tissues in postmenopausal women. The relationship between scattering and tissue composition will support improved scattering models and technologies to enhance intraoperative optical margin assessment.
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http://dx.doi.org/10.1117/1.JBO.21.6.066014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914603PMC
June 2016

Rapid staining and imaging of subnuclear features to differentiate between malignant and benign breast tissues at a point-of-care setting.

J Cancer Res Clin Oncol 2016 Jul 22;142(7):1475-86. Epub 2016 Apr 22.

Department of Biomedical Engineering, Duke University, 136 Hudson Hall Box 90281, Durham, NC, 27708, USA.

Purpose: Histopathology is the clinical standard for tissue diagnosis; however, it requires tissue processing, laboratory personnel and infrastructure, and a highly trained pathologist to diagnose the tissue. Optical microscopy can provide real-time diagnosis, which could be used to inform the management of breast cancer. The goal of this work is to obtain images of tissue morphology through fluorescence microscopy and vital fluorescent stains and to develop a strategy to segment and quantify breast tissue features in order to enable automated tissue diagnosis.

Methods: We combined acriflavine staining, fluorescence microscopy, and a technique called sparse component analysis to segment nuclei and nucleoli, which are collectively referred to as acriflavine positive features (APFs). A series of variables, which included the density, area fraction, diameter, and spacing of APFs, were quantified from images taken from clinical core needle breast biopsies and used to create a multivariate classification model. The model was developed using a training data set and validated using an independent testing data set.

Results: The top performing classification model included the density and area fraction of smaller APFs (those less than 7 µm in diameter, which likely correspond to stained nucleoli).When applied to the independent testing set composed of 25 biopsy panels, the model achieved a sensitivity of 82 %, a specificity of 79 %, and an overall accuracy of 80 %.

Conclusions: These results indicate that our quantitative microscopy toolbox is a potentially viable approach for detecting the presence of malignancy in clinical core needle breast biopsies.
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http://dx.doi.org/10.1007/s00432-016-2165-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900949PMC
July 2016

A Fluorescence-Guided Laser Ablation System for Removal of Residual Cancer in a Mouse Model of Soft Tissue Sarcoma.

Theranostics 2016 1;6(2):155-66. Epub 2016 Jan 1.

6. Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina.

The treatment of soft tissue sarcoma (STS) generally involves tumor excision with a wide margin. Although advances in fluorescence imaging make real-time detection of cancer possible, removal is limited by the precision of the human eye and hand. Here, we describe a novel pulsed Nd:YAG laser ablation system that, when used in conjunction with a previously described molecular imaging system, can identify and ablate cancer in vivo. Mice with primary STS were injected with the protease-activatable probe LUM015 to label tumors. Resected tissues from the mice were then imaged and treated with the laser using the paired fluorescence-imaging/ laser ablation device, generating ablation clefts with sub-millimeter precision and minimal underlying tissue damage. Laser ablation was guided by fluorescence to target tumor tissues, avoiding normal structures. The selective ablation of tumor implants in vivo improved recurrence-free survival after tumor resection in a cohort of 14 mice compared to 12 mice that received no ablative therapy. This prototype system has the potential to be modified so that it can be used during surgery to improve recurrence-free survival in patients with cancer.
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http://dx.doi.org/10.7150/thno.13536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729765PMC
January 2017

Structured Illumination Microscopy and a Quantitative Image Analysis for the Detection of Positive Margins in a Pre-Clinical Genetically Engineered Mouse Model of Sarcoma.

PLoS One 2016 22;11(1):e0147006. Epub 2016 Jan 22.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

Intraoperative assessment of surgical margins is critical to ensuring residual tumor does not remain in a patient. Previously, we developed a fluorescence structured illumination microscope (SIM) system with a single-shot field of view (FOV) of 2.1 × 1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 μm). The goal of this study was to test the utility of this technology for the detection of residual disease in a genetically engineered mouse model of sarcoma. Primary soft tissue sarcomas were generated in the hindlimb and after the tumor was surgically removed, the relevant margin was stained with acridine orange (AO), a vital stain that brightly stains cell nuclei and fibrous tissues. The tissues were imaged with the SIM system with the primary goal of visualizing fluorescent features from tumor nuclei. Given the heterogeneity of the background tissue (presence of adipose tissue and muscle), an algorithm known as maximally stable extremal regions (MSER) was optimized and applied to the images to specifically segment nuclear features. A logistic regression model was used to classify a tissue site as positive or negative by calculating area fraction and shape of the segmented features that were present and the resulting receiver operator curve (ROC) was generated by varying the probability threshold. Based on the ROC curves, the model was able to classify tumor and normal tissue with 77% sensitivity and 81% specificity (Youden's index). For an unbiased measure of the model performance, it was applied to a separate validation dataset that resulted in 73% sensitivity and 80% specificity. When this approach was applied to representative whole margins, for a tumor probability threshold of 50%, only 1.2% of all regions from the negative margin exceeded this threshold, while over 14.8% of all regions from the positive margin exceeded this threshold.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147006PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4723137PMC
July 2016

Design of a Novel Low Cost Point of Care Tampon (POCkeT) Colposcope for Use in Resource Limited Settings.

PLoS One 2015 2;10(9):e0135869. Epub 2015 Sep 2.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America; Center for Global Women's Health Technologies, Duke University, Durham, North Carolina, United States of America; Duke Global Health Institute, Duke University, Durham, North Carolina, United States of America.

Introduction: Current guidelines by WHO for cervical cancer screening in low- and middle-income countries involves visual inspection with acetic acid (VIA) of the cervix, followed by treatment during the same visit or a subsequent visit with cryotherapy if a suspicious lesion is found. Implementation of these guidelines is hampered by a lack of: trained health workers, reliable technology, and access to screening facilities. A low cost ultra-portable Point of Care Tampon based digital colposcope (POCkeT Colposcope) for use at the community level setting, which has the unique form factor of a tampon, can be inserted into the vagina to capture images of the cervix, which are on par with that of a state of the art colposcope, at a fraction of the cost. A repository of images to be compiled that can be used to empower front line workers to become more effective through virtual dynamic training. By task shifting to the community setting, this technology could potentially provide significantly greater cervical screening access to where the most vulnerable women live. The POCkeT Colposcope's concentric LED ring provides comparable white and green field illumination at a fraction of the electrical power required in commercial colposcopes. Evaluation with standard optical imaging targets to assess the POCkeT Colposcope against the state of the art digital colposcope and other VIAM technologies.

Results: Our POCkeT Colposcope has comparable resolving power, color reproduction accuracy, minimal lens distortion, and illumination when compared to commercially available colposcopes. In vitro and pilot in vivo imaging results are promising with our POCkeT Colposcope capturing comparable quality images to commercial systems.

Conclusion: The POCkeT Colposcope is capable of capturing images suitable for cervical lesion analysis. Our portable low cost system could potentially increase access to cervical cancer screening in limited resource settings through task shifting to community health workers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135869PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4557989PMC
May 2016

A quantitative microscopic approach to predict local recurrence based on in vivo intraoperative imaging of sarcoma tumor margins.

Int J Cancer 2015 Nov 3;137(10):2403-12. Epub 2015 Jun 3.

Department of Biomedical Engineering, Duke University, Durham, North Carolina.

The goal of resection of soft tissue sarcomas located in the extremity is to preserve limb function while completely excising the tumor with a margin of normal tissue. With surgery alone, one-third of patients with soft tissue sarcoma of the extremity will have local recurrence due to microscopic residual disease in the tumor bed. Currently, a limited number of intraoperative pathology-based techniques are used to assess margin status; however, few have been widely adopted due to sampling error and time constraints. To aid in intraoperative diagnosis, we developed a quantitative optical microscopy toolbox, which includes acriflavine staining, fluorescence microscopy, and analytic techniques called sparse component analysis and circle transform to yield quantitative diagnosis of tumor margins. A series of variables were quantified from images of resected primary sarcomas and used to optimize a multivariate model. The sensitivity and specificity for differentiating positive from negative ex vivo resected tumor margins was 82 and 75%. The utility of this approach was tested by imaging the in vivo tumor cavities from 34 mice after resection of a sarcoma with local recurrence as a bench mark. When applied prospectively to images from the tumor cavity, the sensitivity and specificity for differentiating local recurrence was 78 and 82%. For comparison, if pathology was used to predict local recurrence in this data set, it would achieve a sensitivity of 29% and a specificity of 71%. These results indicate a robust approach for detecting microscopic residual disease, which is an effective predictor of local recurrence.
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http://dx.doi.org/10.1002/ijc.29611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575838PMC
November 2015

Delivery-corrected imaging of fluorescently-labeled glucose reveals distinct metabolic phenotypes in murine breast cancer.

PLoS One 2014 19;9(12):e115529. Epub 2014 Dec 19.

Department of Biomedical Engineering, Duke University, Durham, NC, United States of America.

When monitoring response to cancer therapy, it is important to differentiate changes in glucose tracer uptake caused by altered delivery versus a true metabolic shift. Here, we propose an optical imaging method to quantify glucose uptake and correct for in vivo delivery effects. Glucose uptake was measured using a fluorescent D-glucose derivative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-D-glucose (2-NBDG) in mice implanted with dorsal skin flap window chambers. Additionally, vascular oxygenation (SO2) was calculated using only endogenous hemoglobin contrast. Results showed that the delivery factor proposed for correction, "RD", reported on red blood cell velocity and injected 2-NBDG dose. Delivery-corrected 2-NBDG uptake (2-NBDG60/RD) inversely correlated with blood glucose in normal tissue, indicating sensitivity to glucose demand. We further applied our method in metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. The ratio 2-NBDG60/RD was increased in 4T1 tumors relative to 4T07 tumors yet average SO2 was comparable, suggesting a shift toward a "Warburgian" (aerobic glycolysis) metabolism in the metastatic 4T1 line. In heterogeneous regions of both 4T1 and 4T07, 2-NBDG60/RD increased slightly but significantly as vascular oxygenation decreased, indicative of the Pasteur effect in both tumors. These data demonstrate the utility of delivery-corrected 2-NBDG and vascular oxygenation imaging for differentiating metabolic phenotypes in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115529PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272314PMC
December 2015

Chromophore based analyses of steady-state diffuse reflectance spectroscopy: current status and perspectives for clinical adoption.

J Biophotonics 2015 Jan 23;8(1-2):9-24. Epub 2014 Apr 23.

Philips Research, Minimally Invasive Healthcare Department, HTC 34, The Netherlands.

Diffuse reflectance spectroscopy is a rapidly growing technology in the biophotonics community where it has shown promise in its ability to classify different tissues. In the steady-state domain a wide spectrum of clinical applications is supported with this technology ranging from diagnostic to guided interventions. Diffuse reflectance spectra provide a wealth of information about tissue composition; however, extracting biologically relevant information from the spectra in terms of chromophores may be more useful to gain acceptance into the clinical community. The chromophores that absorb light in the visible and near infrared wavelengths can provide information about tissue composition. The key characteristics of these chromophores and their relevance in different organs and clinical applications is the focus of this review, along with translating their use to the clinic.
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http://dx.doi.org/10.1002/jbio.201300198DOI Listing
January 2015

Rapid determination of oxygen saturation and vascularity for cancer detection.

PLoS One 2013 16;8(12):e82977. Epub 2013 Dec 16.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

A rapid heuristic ratiometric analysis for estimating tissue hemoglobin concentration and oxygen saturation from measured tissue diffuse reflectance spectra is presented. The analysis was validated in tissue-mimicking phantoms and applied to clinical measurements in head and neck, cervical and breast tissues. The analysis works in two steps. First, a linear equation that translates the ratio of the diffuse reflectance at 584 nm and 545 nm to estimate the tissue hemoglobin concentration using a Monte Carlo-based lookup table was developed. This equation is independent of tissue scattering and oxygen saturation. Second, the oxygen saturation was estimated using non-linear logistic equations that translate the ratio of the diffuse reflectance spectra at 539 nm to 545 nm into the tissue oxygen saturation. Correlations coefficients of 0.89 (0.86), 0.77 (0.71) and 0.69 (0.43) were obtained for the tissue hemoglobin concentration (oxygen saturation) values extracted using the full spectral Monte Carlo and the ratiometric analysis, for clinical measurements in head and neck, breast and cervical tissues, respectively. The ratiometric analysis was more than 4000 times faster than the inverse Monte Carlo analysis for estimating tissue hemoglobin concentration and oxygen saturation in simulated phantom experiments. In addition, the discriminatory power of the two analyses was similar. These results show the potential of such empirical tools to rapidly estimate tissue hemoglobin in real-time spectral imaging applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082977PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3865147PMC
October 2014

Optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells.

Chem Biol 2013 Sep 12;20(9):1187-97. Epub 2013 Sep 12.

Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA.

Inhibitors of heat-shock protein 90 (Hsp90) have demonstrated an unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained, but could be influenced by ectopically expressed Hsp90 in tumors. In this work, we synthesized Hsp90 inhibitors that can carry optical or radioiodinated probes via a polyethyleneglycol tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein occurs at the plasma membrane. In mice, we observed exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors that target ectopic Hsp90 can be used to detect breast cancer malignancies through noninvasive imaging.
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http://dx.doi.org/10.1016/j.chembiol.2013.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815696PMC
September 2013

Optical spectral surveillance of breast tissue landscapes for detection of residual disease in breast tumor margins.

PLoS One 2013 26;8(7):e69906. Epub 2013 Jul 26.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, United States of America.

We demonstrate a strategy to "sense" the micro-morphology of a breast tumor margin over a wide field of view by creating quantitative hyperspectral maps of the tissue optical properties (absorption and scattering), where each voxel can be deconstructed to provide information on the underlying histology. Information about the underlying tissue histology is encoded in the quantitative spectral information (in the visible wavelength range), and residual carcinoma is detected as a shift in the histological landscape to one with less fat and higher glandular content. To demonstrate this strategy, fully intact, fresh lumpectomy specimens (n = 88) from 70 patients were imaged intra-operatively. The ability of spectral imaging to sense changes in histology over large imaging areas was determined using inter-patient mammographic breast density (MBD) variation in cancer-free tissues as a model system. We discovered that increased MBD was associated with higher baseline β-carotene concentrations (p = 0.066) and higher scattering coefficients (p = 0.007) as measured by spectral imaging, and a trend toward decreased adipocyte size and increased adipocyte density as measured by histological examination in BMI-matched patients. The ability of spectral imaging to detect cancer intra-operatively was demonstrated when MBD-specific breast characteristics were considered. Specifically, the ratio of β-carotene concentration to the light scattering coefficient can report on the relative amount of fat to glandular density at the tissue surface to determine positive margin status, when baseline differences in these parameters between patients with low and high MBD are taken into account by the appropriate selection of threshold values. When MBD was included as a variable a priori, the device was estimated to have a sensitivity of 74% and a specificity of 86% in detecting close or positive margins, regardless of tumor type. Superior performance was demonstrated in high MBD tissue, a population that typically has a higher percentage of involved margins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069906PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724737PMC
April 2014

Optimization of a widefield structured illumination microscope for non-destructive assessment and quantification of nuclear features in tumor margins of a primary mouse model of sarcoma.

PLoS One 2013 23;8(7):e68868. Epub 2013 Jul 23.

Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1 × 1.6 mm (3.4 mm(2)) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm(-1) used in conjunction with a 4 × 0.1 NA objective (v=0.165). This yielded an optical section thickness of 128 µm and an 8.9 × contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068868PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720887PMC
March 2014
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