The Second Britton Chance International Symposium on Metabolic Imaging and Spectroscopy

(in alphabetical order)

A B C D F G H I J K L M O R S V W Z

Cerenkov imaging of breast cancer cells with different metastatic potential
Alejandro D. Arroyo, Anatoliy V. Popov, E. James Delikatny
Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

This project focuses on the development of functional bioactivatable molecules to image tumor metabolic features using Cerenkov imaging. Resazurin (RA_ox), also known as Alamar Blue, is a redox sensor and viability dye. NAD(P)H dehydrogenases and reducing agents can reduce RAox into resorufin (RA_red), a highly fluorescent probe. Using electrophilic fluorination, monofluororesazurin (MFRA_ox) was synthesized [1]. MFRA_ox was validated to have similar redox monitoring properties as RA_ox. Use of 18F-labeled MFRA_ox allows for localization of the probe and biodistribution studies by PET imaging, and assessment of metabolic signatures by Cerenkov imaging. Cerenkov radiation and the fluorescence by RA_red can produce Cerenkov Radiation Energy Transfer (CRET), an emission closer to the near infrared (NIR) window, better suited for in vivo imaging. Cerenkov imaging of MFRA confirmed the ability to generate CRET which could be used to differentiate between MFRA_red and MFRA_ox with an emission ratio of 3.5:1 at 640 nm. The breast cancer cell lines chosen for this project have different metastatic potentials: MDA-MB-231, a triple-negative human breast cancer, and 4175-Luc+, a highly metastatic MDA-MB-231 variant isolated from murine lung metastasis [2]. Using fluorescence, it was determined that 4175-Luc+ cells reduced MFRA_ox significantly faster than MDA-MB-231 cells. This difference in reduction rate could be correlated to metastatic potential. Cerenkov imaging after intratumoral injections of ¹⁸F-FDG-MFRA_ox showed faster reduction of the probe in 4175-Luc+ tumors than in MDA-MB-231. This was further confirmed after tumor excision. The faster reduction of MFRA_ox in 4175-Luc+ cells and tumors can be correlated with increased glycolytic activity, determined by higher glucose consumption and lactate production. Since deregulated cellular energetics constitute one of the Hallmarks of Cancer [3], MFRA_ox could be used to probe energy imbalance in cancer cells, reductive environment and metastatic potential of tumor

References:
1. Kachur AV, Arroyo AD, et al. (2015) Synthesis of F-18 labeled resazurin by direct electrophilic fluorination. J Fluor Chem. 178:136-141.
2. Minn, AJ, et. al. (2005) Genes that mediate breast cancer metastasis to lung. Nature 436(7050):518–524.
3. Hanahan D, Weinberg RA. (2011) Hallmarks of Cancer: The Next Generation. Cell 440(5): 646-674.

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Detection of cerebral Nicotinamide adenine dinucleotide (NAD⁺) in humans at 7T MRI
Puneet Bagga¹, Kevin D’Aquilla¹, Neil Wilson¹, Mark Elliott¹, Mohammad Haris², Ravi Prakash Reddy Nanga¹, John Detre³, Hari Hariharan¹, and Ravinder Reddy^1
1 Center for Magnetic Resonance and Optical Imaging, Department of Radiology, University of Pennsylvania, Philadelphia, PA; ² Research Branch, Sidra Medical and Research Center, Doha, Qatar; ³Department of Neurology, University of Pennsylvania, Philadelphia, PA

Nicotinamide adenine dinucleotide (NAD⁺) plays a vital role in the cellular metabolism as a coenzyme for electron transfer enzymes in addition to the reduction-oxidation reactions have the involvement of NAD⁺ and NADH^1,2. Thus, in vivo monitoring of NAD⁺ levels can be used to study the oxidative stress levels related to various physiological conditions. Currently, the in vivo detection of NAD species (NAD⁺+NADH) is performed mostly via ³¹P MR spectroscopy. However, ³¹P MRS is limited by lower spectral resolution making it difficult to resolve the oxidized and reduced forms of NADP species3. Very recently, 1H MR spectroscopy in a slice using a surface coil has been shown to detect NAD⁺ in the brain^4,5. In the current study, we present a new sequence and method to detect single voxel localized 1H MRS detection of NAD+ from the human brain at 7T using a volume coil. The pulse sequence uses outer volume suppression (OVS) block with 6 saturation pulses with spatial bandwidth 10cm and gap of 2cm around all the three dimensions of the MRS voxel. We use two OVS blocks separated by 30 ms followed by a spectrally selective self-refocused E-BURP1 90° pulse and 3 narrow band (800 Hz) spatially selective refocusing 180° Shinnar-Le Roux pulses for localization⁶. The 90° pulse is 7ms long and bandwidth 2 ppm (600 Hz at 7T). The spectrally selective pulse and the 180° pulses are centered at 9.1 ppm for NAD and 4.7 ppm for water. Refocusing pulses that are spatially selective for localization are based on Shinnar-Le Roux pulse design and are 3.2ms long with a bandwidth of 800 Hz giving an overall TE of 19.3ms. Water reference acquisitions were done with TR = 8s and 4 avgs and NAD⁺ acquisitions were done with TR=1s and 512 avgs. For NAD⁺ quantification, the spectrum in the range of 9 to 9.5 ppm was fitted to a sum of 2 Lorentzians and from the integral of the 9.34 ppm peak, NAD⁺ concentration was calculated by normalization with the water integral (assuming 80% water content in the brain). The non-overlapping NAD⁺ H2 resonance at 9.35 ppm was quantified to be ~0.26 mM, which is in agreement with the reported literature^3,4,5.

REFERENCES:
1. Stein LR and Imai S (2012) Trends Endocrinol Metab 23:420-438
2. Bai P and Canto C (2012) Cell Metab 16:290-295
3. Lu M et al (2014) Magn Res Med 71:1959-1972
4. de Graaf RA and Behar KL (2014) NMR Biomed 27:802-809
5. de Graaf RA et al (2016) Magn Reson Med DOI:10.1002/mrm.26465
6. Geen H and Freeman R (1991) J Magn Reson 93:93-141; 23:420-38

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Bergonzi, Karla M.

Measuring brain connectivity during acute stroke at the bedside using High-Density Diffuse Optical Tomography
Karla M. Bergonzi^1,2, Adam T. Eggebrecht¹, Broc A. Burke³, Andrew K. Fishell⁴, Tracy M. Burns-Yocum¹, Silvina L. Ferradal⁵, Hamid Dehghani⁶, Ben J. Palanca³, Gyan Kumar⁷, Rajat Dhar⁷, Jin-Moo Lee^1,2,7, and Joseph P. Culver^1,2,4,8
1Department of Radiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA ²Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, USA ³Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA ⁴Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA ⁵Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115, USA ⁶School of Computer Science, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK ⁷Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110, USA ⁸Department of Physics, Washington University in St. Louis, St. Louis, Missouri 63130, USA

Ischemic stroke, which presents with the sudden onset of neurological deficits, triggers a complex cascade of events including anoxic depolarization, excitotoxicity, spreading depression, and, in some cases, reperfusion. During the first 72 hours, wherein primary and secondary brain injury evolves rapidly and therapeutic interventions aim to preserve viable brain tissue from secondary damage, early detection of neurological deterioration is essential, and therefore, close neurological monitoring is critical. Treatment during early stroke can increase survival rates and reduce life-altering disabilities. Bedside functional neuroimaging has tremendous potential as a diagnostic and prognostic tool to improve critical care during this period of neurologic instability. We have developed a High-Density Diffuse Optical Tomography system (HD-DOT) that images brain activity at the bedside without interrupting clinical care. We show that HD-DOT is sensitive to altered brain function within 72 hours of ischemic stroke onset. To assess brain function, we developed a functional connectivity-based metric that compares the brain function of a stroke patient at rest to that of a healthy population average. Our results demonstrate that HD-DOT can differentiate between healthy subjects and stroke patients (p<1E-5) and that the degree of disruption in brain connectivity measured with HD-DOT is correlated with the NIHSS (n=13, r = -0.81, R²=0.66, p=7.4E-4), a clinical measure of stroke-induced functional deficit.

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New approach in monitoring redox ratio of cancer cells by FLIRR
Ruofan Cao, Horst Wallrabe, Ammasi Periasamy
The W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA, USA. Departments of Biology, University of Virginia, Charlottesville, VA, USA.

Redox state changes, increased glycolysis and defective mitochondrial oxidative phosphorylation (OXPHOS) are hallmarks of cancer pathology which can be monitored by following the intensities of the co-enzymes FAD and NAD(P)H (and their ratio), pioneered by Britton Chance. Unfortunately, light scattering and differential absorption of light -especially in tissue and deep specimens -makes intensity-based methods problematic or unusable. Our newly developed assay is based entirely on fluorescence lifetime imaging (FLIM) parameters, which are independent of excitation intensity. The fluorescence lifetime redox ratio (FLIRR) is based on NAD(P)H-a2%/FAD-a1% using a two-photon TSCP FLIM imaging system. Two-photon excitation is applied to achieve better non-invasive penetration with reduced toxicity. Furthermore, FLIRR is analyzed in segmented cells, thresholded by small Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis. Hundreds of data points demonstrate heterogeneous response to treatment as measured by FLIRR, which subsequently are categorized into different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This 2-photon FLIRR method provides an intensity independent reading of redox states to detect subtle differences in deeper cellular and tissue responses.

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Cao, Xu

Feasibility of Label-free cerebrovascular imaging using SWIR Cherenkov emission induced by external radiation
Xu Cao^1,3, Mengyu Jia^1,2, Petr Bruza^1,2, Shudong Jiang^1,2, and Brian W. Pogue^1,2
1Thayer School of Engineering; ²Medical School, Dartmouth College, Hanover, NH-03755; ³ Engineering Research Center of Molecular and Neuro Imaging of the Ministry of Education & School of Life Science and Technology, Xidian University, Xi’an, Shaanxi 710071, China

In the past several years, the results from many studies demonstrated that short-wave infrared (SWIR) fluorescence imaging can provide higher spatial resolution for imaging cerebrovasculature of mice in vivo (comparing to the fluorescence imaging in visible and/or near infrared wavelength range), due to the reduction of photon scattering in SWIR spectral region. In contrast to these previous studies with the needs of injection of fluorescence probe, Cherenkov emission induced by megavolt X-ray beam can be harnessed for label-free tissue microenvironment imaging. Due to the camera wavelength limitation, the previous Cherenkov imaging were focused on imaging Cherenkov emission in the visible and NIR wavelength region (400nm-800nm). For understanding whether the Cherenkov emission imaging can be carried out in SWIR wavelength region (900nm-1400nm), the spectrum of SWIR Cherenkov emission was measured based on a system containing a SWIR camera and a fiber bundles with the effective diameter of 5mm. The spectrum obtained with this system is in great agreement with that calculated by Frank-Tamm theory. The results of the series phantom studies show that the intensity of SWIR Cherenkov emission is linearly correlated with the radiation dose of megavolt X-ray beams, and the penetration depth of SWIR Cherenkov emission can reach to 5cm. SWIR Cherenkov emission image of a mouse irradiated by megavolt electron beams was also obtained in vivo. These results demonstrated the high potential to use SWIR Cherenkov emission for label-free cerebrovascular imaging.

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Caporale, Alessandra

Vessel-wall MRI detects impaired flow-mediated dilation after e-cigarette vaping
Alessandra Caporale, Michael Langham, Felix W Wehrli
Department of Radiology, University of Pennsylvania, Philadelphia, PA

SYNOPSIS: The rapid rise in the popularity of the electronic-cigarette (e-cig) is an unsettling trend given the limited knowledge on long-term health effects of aerosol inhalation. Using a rapid vessel-wall MRI technique we observed impaired FMD from the e-cig aerosol inhalation.
BACKGROUND AND PURPOSE: Brachial artery flow-mediated dilation (FMD) has been used as a research tool for quantifying vascular reactivity as a surrogate marker of endothelial function¹. FMD is caused by transient increase in blood flow due to reduced microvascular resistance during hyperemia following induced ischemia. Recent studies indicate that e-cigarette smoking leads to FMD reduction comparable to conventional cigarette with the same amount of nicotine². Our aim was to investigate acute effects of the nicotine-free e-cigarette aerosol on femoral artery vasodilation.
METHODS: Reactive hyperemia in femoral artery was elicited by 5 minutes of cuff occlusion (D.E. Hokanson Inc, Bellevue, WA). With a rapid vessel-wall imaging technique based on 3D steady-state-free-precession-echo^3,4, axial images of the superficial femoral artery were captured at rest and at 60, 90 and 120 seconds after cuff deflation (accounting for subject-dependent peak dilation time5). Luminal femoral artery FMD was computed as the relative percentage change between the cross-sectional area at rest and peak dilation, doubling the sensitivity to FMD compared to the conventional ultrasound-based method¹. MRI was performed at 3T (Siemens Trio scanner), using an 8-channel transmit/receive extremity coil, before and after nicotine-free e-cigarette smoking in three healthy non-smokers (females, mean age:23±1years). In 10 subjects FMD was quantified without e-cigarette challenge to determine within-subject variability.
RESULTS: FMD measured before e-cigarette vaping in the three subjects was 14%, 21%, 7%, decreasing to 7%, 6%, 0%, respectively. The mean difference in FMD between pre- and post- e-cigarette challenge was 9.7±4.6%; the intra-subject variability in FMD in test subjects was 0.31±3.8%.
DISCUSSION AND CONCLUSIONS: In three subjects luminal femoral artery FMD was reduced after an e-cigarette aerosol inhalation challenge, probably in response to increased oxidative stress caused by ultrafine particles and free radical inhalation and subsequent translocation into the bloodstream^6-9. These initial observations, while encouraging, will need verification in appropriately powered studies.

 

Figure 1. Flow mediated dilation (FMD): a) High resolution SSFP-echo image of superficial femoral artery, showing the slice of interest. b) Magnification of femoral artery shown in a), and schematic of the acquisition protocol: A0 represents the cross-sectional area at rest; A60s, A90s, A120s, represent 3 successive acquisitions during artery lumen dilation, at 60, 90, 120 s, respectively, from cuff deflation. c) Comparison between flow-mediated dilation in a representative subject before and after smoking e-cigarette aerosol, showing FMD impairment.

REFERENCES:
1. Corretti MC, Anderson TJ, Benjamin EJ, et al. (2002) Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. Journal of the American College of Cardiology. 39(2):257-65.
2. Carnevale R, Sciarretta S, Violi F, et al. (2016) Acute impact of tobacco vs electronic cigarette smoking on oxidative stress and vascular function. Chest. 150(3):606-612.
3. Langham MC, Desjardins B, Englund EK, et al. (2016) Rapid high-resolution, self-registered, dual lumen-contrast MRI method for vessel-wall assessment in peripheral artery disease: A preliminary investigation. Academic radiology 23(4):457-467.
4. Langham MC, Li C, Englund EK, et al. (2013) Vessel-wall imaging and quantification of flow-mediated dilation using water-selective 3D SSFP-echo. Journal of Cardiovascular Magnetic Resonance 15(1):100.
5. Black MA, Cable NT, Thijssen DH, Green DJ. (2008) Importance of measuring the time course of flow-mediated dilatation in humans. Hypertension 51(2):203-210.
6. Goel R, Durand E, Trushin N, et al. (2015) Highly reactive free radicals in electronic cigarette aerosols. Chemical Research in Toxicology 28(9):1675-7.
7. Nakane H. (2012) Translocation of particles deposited in the respiratory system: a systematic review and statistical analysis. Environmental Health and Preventive Medicine. 17(4):263.
8. Rundell KW, Hoffman JR, Caviston R, et al. (2007) Inhalation of ultrafine and fine particulate matter disrupts systemic vascular function. Inhalation Toxicology. 19(2):133-140.
9. Zhang Y, Sumner W, Chen D-R. (2012) In vitro particle size distributions in electronic and conventional cigarette aerosols suggest comparable deposition patterns. Nicotine & Tobacco Research 15(2):501-508.

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Chawla, Sanjeev

Three-dimensional echo-planar spectroscopic imaging for differentiation of true progression from pseudo-progression in patients with glioblastoma
Sanjeev Chawla¹, Gaurav Verma¹, Sumei Wang¹, MacLean Nasrallah², Sulaiman Sheriff³, Arati Desai⁴, Steven Brem⁵, Donald M. O’Rourke⁵, Ronald L. Wolf¹, Andrew A. Maudsley³, Harish Poptani^1,6, Suyash Mohan¹
Departments of ¹Radiology, ²Pathology and Lab Medicine, ⁴Hematology-Oncology, ⁵Neurosurgery, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA; ³Department of Radiology, University of Miami, Miami, FL, ⁶Department of Cellular and Molecular Physiology, University of Liverpool, Liverpool, UK

Accurate differentiation of true progression (TP) from pseudo-progression (PsP) in patients with glioblastomas (GBMs) is essential for planning adequate treatment and for estimating clinical outcome measures and future prognosis. The purpose of this study was to investigate the utility of three-dimensional echo-planar spectroscopic imaging (3D-EPSI) in distinguishing TP from PsP in GBM patients. A cohort of 27 patients with GBM demonstrating enhancing lesions within 6-months after completion of concurrent chemo-radiation therapy were included. Of these, 18 were subsequently classified as TP and 9 as PsP based on histological features or follow-up MRI studies. Parametric maps of choline/creatine (Cho/Cr) and Cho/N-acetylaspartate (Cho/NAA) were computed and co-registered with post-contrast T1-weighted and FLAIR images. All lesions were segmented into contrast enhancing (CER), immediate peritumoral (IPR), and distal peritumoral regions (DPR). For each region, median values of Cho/Cr and Cho/NAA ratios were normalized to corresponding metabolite ratios from contralateral normal parenchyma and compared between TP and PsP groups using Mann-Whitney-U tests. A probabilistic (p) value of less than 0.05 was considered significant. Logistic regression analyses were performed to obtain the best model in distinguishing TP from PsP. Significantly higher Cho/NAA was observed from CER (2.69±1.00 vs. 1.56±0.51, p=0.003), IPR (2.31±0.92 vs. 1.53±0.56, p=0.030); and DPR (1.80±0.68 vs. 1.19±0.28, p=0.035) regions in TP patients compared to those with PsP. Additionally, significantly elevated Cho/Cr (1.74±0.44 vs. 1.34±0.26, p=0.023) from CER was observed in TP compared to PsP. When these parameters were incorporated in multivariate regression analyses, a discriminatory model with a sensitivity of 94% and a specificity of 87% was observed in distinguishing TP from PsP. Leave-one-out cross-validation test revealed that 92.3% of patients were correctly classified as TP or PsP using the multivariate logistic regression model. These results indicate the utility of 3D-EPSI in differentiating TP from PsP with high sensitivity and specificity.

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Chong, Sanghoon

Novel approach to spatial frequency domain fluorescence diffuse optical tomography for tumor imaging
Sanghoon Chong¹, Vadim A. Markel², Ashwin B. Parthasarathy³ Yi Hong Ong^1,4, Frank A. Moscatelli⁵, and Arjun G. Yodh^1
1Department of Physics and Astronomy, University of Pennsylvania; ²Division of Radiology and Bioengineering, University of Pennsylvania; ³Department of Electrical Engineering, University of South Florida; ⁴Department of Radiation Oncology, Hospital of the University of Pennsylvania; ⁵Department of Physics, New York University

Fluorescence image guided surgery has become one of the most important modalities for tumor resection surgery since its first emergence. Accordingly, the optical community studied fluorescence imaging technique to assist surgeons to make a decision on tumor margin, especially in order to acquire tomographic image reconstruction. In the same effort, we have critically studied singular value decomposition (SVD) based analytic inversion technique for fluorescence diffuse optical tomography under spatially structured illumination. During this investigation, we learned that employing the full scale of the SVD-based analytic inversion is intrinsically vulnerable to high frequency noise. As a result, resolution of depth and lateral margin of fluorescent contrast target are extremely difficult to be specified. Thereby, we propose a novel approach to ameliorate the inherent weakness of the SVD-based analytic inversion technique in spatial frequency domain imaging based on our discovery; depth of the contrast target can be accurately acquired in attempt to reconstruct DC Fourier component of fluorophore concentration in the contrast target at each depth. Based on this discovery, we developed two-step reconstruction technique in order to acquire accurate depth sensitivity and lateral margin. The first step is to (1) reconstruct DC component of concentration at each depth and create a concentration profile depending on depth. The maximum concentration point in the profile indicates the accurate target depth. Next, (2) based on the correct depth information from step (1), we can define a thin slice at the recovered depth, and then reconstruct the target within the thin slab to determine its lateral margin. We present the theory of DC Fourier component reconstruction technique as well as reconstruction examples using inverse crime simulation data. The reconstruction examples include results from (1) full Fourier spectrum of contrast and (2) DC Fourier component of contrast for comparison.

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Cochran, Jeffrey M.

Tumor oxygenation predicts response to neoadjuvant chemotherapy within 10 days
Jeffrey M. Cochran¹, David R. Busch², Anaïs Leproux³, Bruce J. Tromberg³, Arjun G. Yodh^3
1Department of Physics and Astronomy, University of Pennsylvania; ²Department of Anesthesiology and Pain Management, University of Texas Southwestern; ³Beckman Laser Institute and Medical Clinic, University of California, Irvine

Neoadjuvant chemotherapy (NAC) is a common treatment paradigm for locally advanced breast cancer. Ideally, assessment of treatment efficacy would occur as early as possible during the course of the 3-6 month therapy regimen and predict pathologic complete response (pCR), a surrogate clinical endpoint for 5-year survival. We introduce and demonstrate a new approach for predicting pCR within 10 days of initiating NAC. The method uses the non-invasive, bedside diffuse optical spectroscopic imaging (DOSI) technology combined with logistic regression modeling. Tumor and normal tissue physiological properties were measured longitudinally throughout the course of NAC in 33 patients enrolled in the American College of Radiology Imaging Network multi-site breast cancer DOSI trial (ACRIN-6691). An image analysis scheme employing z-score normalization to healthy tissue on the tumor-bearing breast produced models with robust predictions. Notably, logistic regression of z-score normalized data using only the tissue oxygen saturation (S_tO₂) measured within 10 days of the initial therapy dose was found to be a significant predictor of pCR (AUC=0.92; 95% CI: 0.82–1). The observation suggests that patients who exhibit convergence of tumor tissue S_tO₂to surrounding tissue S_tO₂ are more likely to achieve pCR. This early predictor of response to therapy occurs prior to reductions in tumor size and could enable individualized, dynamic feedback in the optimization of chemotherapy strategies and patient outcomes in breast cancer.

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Elucidating breast cancer pathophysiology using integrated dynamic DOT and digital breast tomosynthesis
Bin Deng¹, Bernhard B. Zimmermann², Bhawana Singh¹, Qianqian Fang³, Jayne Cormier⁴, Richard H. Moore⁴, Daniel B. Kopans⁴, Mansi A. Saksena⁴, David A. Boas,^1,2, and Stefan A. Carp^1
1Massachusetts General Hospital, Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, MA 02129; ² Boston University, Department of Biomedical Engineering, Boston, MA 02115; ³Northeastern University, Department of Bioengineering, Boston, MA 02115; ⁴Massachusetts General Hospital, Breast Imaging Division, Department of Radiology, Boston, MA 02114

Near-infrared (NIR) diffuse optical tomography (DOT) is emerging as a non-invasive functional imaging method for breast cancer diagnosis and neoadjuvant chemotherapy monitoring. Moreover, dynamic DOT (DDOT) measurements of breast during breath maneuvers, gas inhalation, or mechanical stimulation have shown to offer a novel contrast mechanism that could elucidate tissue pathophysiology. Here, we demonstrated the use of a DDOT apparatus designed for tight integration with commercial digital breast tomosynthesis (DBT) scanners to track hemodynamic changes induced by stepwise mammographic breast compressions. Breast cancer patients presented with either benign or malignant lesions were imaged for 60 seconds under half mammographic force, followed by 60 seconds under full mammographic compression for both breasts. We note a distinctively more pronounced reduction in total hemoglobin concentration in malignant lesions than benign cases across the transition from half to full compression. We conclude that compression-induced changes in breast physiological properties may prove useful in differential diagnosis of breast cancer.

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BP neural network-based reconstruction algorithm for diffuse optical tomography
Jinchao Feng, Qiuwan Sun, Zhe Li, Zhonghua Sun, and Kebin Jia
Faculty of Information Technology, Beijing University of Technology, Beijing, China, 100124

Diffuse optical tomography (DOT) is a promising noninvasive imaging modality capable of providing the functional characteristics of biological tissue by quantifying the optical parameters. The inverse problem in DOT reconstruction are nonlinear, ill-posed and ill-conditioned because of the diffusive nature of scattered light. The conventional reconstruction algorithms for DOT are mostly iterative based methods, which are developed based on Tikhonov regularization. However, the reconstructed DOT images based on Tikhonov regularization are usually over-smoothed and also susceptible to noise to arise artifacts. In addition, the choice of regularization parameter is necessary. Here, a reconstruction algorithm based on back-propagation (BP) neural network is developed for DOT, and its feasibility is evaluated with simulation experiments. Compared to Tikhonov regularization based DOT reconstruction methods, the quantitative accuracy of reconstructed optical properties and spatial resolution are significantly improved with the proposed reconstruction algorithm.

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Feng, Min

Potential biomarker for invasiveness of triple negative breast cancer cells by optical redox imaging
Min Feng, He N. Xu, and Lin Z. Li
Department of Radiology and Britton Chance Laboratory of Redox Imaging, Johnson Research Foundation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104

The invasive and/or metastatic potential of cancer is a significant factor in tumor progression. Invasion and metastasis remain a challenge in cancer research and clinical diagnosis. Optical redox imaging (ORI) technique, based on detecting the endogenous fluorescence signals of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp containing flavin adenine dinucleotide, i.e., FAD), has already been employed in many studies for cancer treatment response, diagnosis or prognosis. NADH, Fp and the redox ratio (Fp/(NADH+Fp) have been shown to be sensitive to mitochondrial energy metabolism of cancer cells. In this study, we aimed to identify an innovative biomarker for differentiating among triple negative breast cancer cells (TNBC) with different invasive potentials by ORI of the cultured cancer cells. Using a fluorescence microscope, we acquired the fluoresce signals of NADH and Fp with proper excitation/emission filters (NADH: 360/455nm; FP: 470/520nm) from three TNBC lines (MDA-MB-231, MDA-MB-436 and MDA-MB-468). The Boyden chamber method was employed to measure the invasive potential (IVP) of the cancer cells. The ORI results showed that the Fp redox ratio significantly differentiated among the three TNBC lines (all pairwise t-test p values < 0.05). The IVP results showed a significant difference among the 3 lines as well. Furthermore, there is a positive correlation between the Fp redox ratio and the IVP value (r=0.76), where the most invasive MDA-MB-231 has the highest Fp/(Fp+NADH) value, and the least invasive MDA-MB-468 has the lowest Fp/(Fp+NADH) value. These data show that the Fp redox ratio has a potential to be a novel biomarker for the prognosis of TNBC in the clinics. 

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Forti, Radrigo

Cerebral optical monitoring during endovascular treatment of stroke
Rodrigo M. Forti^1,2,3, Wesley B. Baker⁴, Scott E. Kasner⁵, Michael T. Mullen⁵, Steven R. Messé⁵, John A. Detre^5,64, W. Andrew Kofke⁷, Rickson C. Mesquita^1,2, Arjun G. Yodh³ and Christopher G. Favilla^5
1Institute of Physics “Gleb Wataghin”, University of Campinas; ² Brazilian Institute of Neuroscience and Neurotechnology; ³Department of Physics & Astronomy, University of Pennsylvania; ⁴Department of Neurology, Children’s Hospital of Philadelphia; ⁵Department of Neurology, University of Pennsylvania; ⁶Department of Radiology, University of Pennsylvania; ⁷Department of Anesthesiology and Critical Care

Optimization of cerebral blood flow (CBF) is critical in the clinical management for many neurological disease states, most notably acute ischemic stroke. Endovascular thrombectomy represents a revolutionary change in the acute treatment of stroke due to large vessel occlusion with demonstrated robust improvements in outcome. Though standard neuroimaging modalities may provide a snapshot of CBF, little is known about the real-time changes on contra- and ipsi-lateral CBF during and after successful recanalization. Diffuse optical techniques, most notably diffuse optical spectroscopy (DOS) and diffuse correlation spectroscopy (DCS), are promising tools for continuous non-invasive monitoring of cerebral hemodynamics. By shining light into the lateral aspect of the forehead, it is possible to measure tissue oxygenation (DOS) and cerebral blood flow (DCS) in the anterior frontal cortex. In this case study, we used a combined DOS-DCS system to monitor frontal lobe hemodynamic changes during endovascular treatment of a patient with acute intracranial carotid occlusion. To account for scalp influences on the optical signal, we employed a pressure modulation algorithm previously developed by our group. Immediately upon recanalization, we found a persistent 120% increase in ipsilateral CBF and a transient 41% increase in contralateral CBF. After 5 mins, contralateral CBF decreased back to baseline, whereas ipsilateral CBF remained elevated for the remaining 25 mins of the experiment. There were no concurrent changes on the extra-cortical blood flow in either hemisphere, during or after recanalization. The measurement of CBF during endovascular treatment with optics further validates diffuse optical techniques as a non-invasive monitor of cerebral hemodynamics. Moreover, the ability to continuously and non-invasively monitor CBF will provide important insight into the cerebral hemodynamics of stroke, and findings may help clinicians better understand complications and clinical changes following successful recanalization.

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Endoscopic redox imaging system for the detection of breast cancer in vivo
Brandon Gaitan¹, Udayakumar Kanniyappan¹, He N. Xu², Qinggong Tang¹, Yi Liu¹, Lin Z. Li², and Yu Chen^1
1Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, ² Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA19104, USA

Breast cancer is the second highest cause of cancer related deaths each year in women. Even though there have been improvements in cancer diagnosis, the mortality rates have not decreased significantly. Excisional biopsy followed by histological examination is currently the gold standard for the diagnosis of cancer, but it can often suffer from high false negative rates due to sampling errors and being subjective. Furthermore, the current method does not provide accurate information for tumor metastatic risk which is needed for individualized treatment. To improve the current situation, new tools need to be developed that not only provide diagnostic but also prognostic information at the time when biopsy is taken. In the 1950-70s, the Chance lab determined that Flavin Adenine Dinucleotide (FAD) and Nicotinamide Adenine Dinucleotide (NADH) could be measured using fluorescence and be used to quantify metabolic parameters in cells and tissue. In the recent years, Lin et al at the University of Pennsylvania have conducted studies in which breast tumors can be differentiated from normal tissue through the fluorescent imaging of FAD and NADH and measuring the tissues redox ratio. The redox ratio of these two molecules(FAD/(FAD+NADH)) has been found to be sensitive to metabolic changes in ex vivo and in vivo tissue. In this study, we have developed a novel needle redox imaging system which can achieve in vivo measurement of redox molecules for clinical cancer diagnosis. This device can fit into the 11G clinical coaxial biopsy needle for real-time imaging during clinical biopsy procedure. Currently we have used liquid and solid phantoms to determine the sensitivity, dynamic range, linearity and resolution of our imaging device. In the future we will perform tests in the clinic on breast cancer patients. Once the device has been validated, it will be an effective tool to assist in breast cancer diagnosis.

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Gillette, Amani

Autofluorescence imaging of oxidative phosphorylation protein knockouts
Amani Gillette^1,2, Peter Favreau², Jarred Rensvold², Ava VanDommelen², David Pagliarini^2,3, Melissa Skala^1,2
1Department of Biomedical Engineering, University of Wisconsin, Madison, WI 53715; ²Morgridge Institute for Research, Madison, WI 53715; ³Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706

Current technologies for cellular metabolomics are complex, costly, and lack single-cell resolution. We propose optical metabolic imaging (OMI) as a tool to noninvasively monitor cellular metabolism on a single-cell level. OMI uses two-photon microscopy and time correlated single photon counting to measure the fluorescence intensity and lifetime of metabolic coenzymes NAD(P)H and FAD. OMI is sensitive to changes in oxidative phosphorylation due to small molecule treatments. However, the biochemical basis of changes in OMI parameters such as redox ratio (NAD(P)H intensity divided by FAD intensity), NAD(P)H lifetime, and FAD lifetime remains unclear. An understanding of these biochemical sources of contrast is needed to interpret OMI studies of drug efficacy, and thus leverage OMI as a tool for drug screening and metabolic research. To address this issue, we profiled 10 CRISPR-mediated knockout human cell lines with single gene deletions corresponding to mitochondrial proteins involved in oxidative phosphorylation. The wild-type and knockout cells were plated at an equal density on 35mm imaging dishes in parallel with additional plates for mass spectrometry analyses. After 48hrs, cells were imaged or collected for mass spectrometry-based metabolomic, proteomic, and lipidomic analyses. OMI of the wild-type and knockout haploid cells yielded statistically significant differences in redox ratio, NAD(P)H and FAD mean lifetimes (ANOVA, p<0.005) that were specific to each knockout. Comparisons between OMI and parallel multi-omic analysis of each knockout are currently underway. This comprehensive dataset will provide a better understanding of the biochemical basis for OMI measurements of oxidative phosphorylation, which could enable robust, single-cell monitoring of metabolic activity.

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Noninvasive continuous optical monitoring of absolute cerebral blood flow in adult human subjects
Lian He¹, Wesley Baker^1,2, Daniel Milej^4,5, Venkaiah Kavuri¹ Rickson C. Mesquita⁷, David Busch^1,3, Mamadou Diop^4,5, Keith St Lawrence^4,5, Ramni Balu², Olivia Amendolia⁶, Francis Quattrone⁶, W. Andrew Kofke^2,6, A. G. Yodh^1,2
1Department of Physics & Astronomy, University of Pennsylvania, Philadelphia, PA, USA; ²Department of Anesthesiology and Critical Care, Hospital of the University of Pennsylvania, Philadelphia, PA, USA; ³Division of Neurology, Children’s Hospital of Philadelphia, PA, USA; ⁴Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada N6A 5C1 ⁵Imaging Division, Lawson Health Research Institute, London, Ontario, Canada, N6A 4V2; ⁶Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, USA; ⁷Department of Cosmic Rays and Chronology, University of Campinas, Campinas, SP, Brazil 13083

We investigate a scheme for noninvasive continuous monitoring of absolute cerebral blood flow (CBF) in adult human patients based on a combination of time-resolved contrast-enhanced near-infrared spectroscopy (DCE-NIRS) and diffuse correlation spectroscopy (DCS).

Continuous CBF is obtained via calibration of the DCS blood flow index (BFI) with absolute CBF obtained by intravenously injected optical contrast agent Indocyanine Green (ICG) at a single time-point, i.e., DCE-NIRS technique. By comparing DCE-NIRS to concurrent DCS measurement, a calibration coefficient (γ) for the cerebral blood flow is determined that permits conversion of DCS BFI to absolute blood flow units at other times during brain injury management. Our study continuously monitored CBF with DCS in 7 adult human patients with traumatic brain injury across multiple days. DCS was calibrated with the ICG bolus DCE-NIRS technique twice daily across our patient population. The concurrent measurements enabled assessment of the stability of ICG calibration coefficients (γ= CBF/BFI) obtained from each patient across single monitoring days.

We found excellent agreement between the two calibration coefficients (R² = 0.80 and slope = 1.02 ± 0.09) across single monitoring days. Additionally, we determined a patient-averaged calibration coefficient (γ=1.27 x 10⁹(ml/100g/min)/(cm²/s)) for DCS monitoring of absolute CBF measured across multiple monitoring days and multiple patients. Significant correlation (R² = 0.89) between CBF obtained with two optical techniques was also observed. Finally, the population-averaged calibration coefficient was applied to a previously published group of adult brain-injured patients thereby taking first steps towards using DCS directly as a measure of absolute CBF. An 8-fold difference was found between the CBF directly measured with DCS and XeCT. In total, the research suggests that the combined DCS and DCE-NIRS method should enable continuous, noninvasive, and quantitative DCS monitoring of absolute CBF at the bedside in the clinic.

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Huang, Tze-ta

Two-channel autofluorescence analysis for oral cancer
Tze-Ta Huang^1,2, Ken-Chung Chen^1,2, Tung-Yiu Wong^1,2 Chih-Yang Chen³, Wang-Ch Chen³ Yi-Chun Chen², Ming-Hsuan Chang⁴, Dong-Yuan Wu⁴, Teng-Yi Huang⁴, Pau-Choo Chung⁵, Jehn-Shyun Huang^1,2
1Division of Oral and Maxillofacial Surgery, Department of stomatology; ²Institute of Oral Medicine, National Cheng-Kung University Medical College and Hospital, Tainan, Taiwan; ³Delta Electronics, Inc; ⁴Institute of Computer and Communication Engineering, National Cheng Kung University, Tainan, Taiwan; ⁵Department of Electrical Engineering, National Cheng Kung University, Tainan, Taiwan

Tissue autofluorescence is a unique feature usable for the optical diagnosis of epithelial carcinogenesis. We created a two-channel autofluorescence test to detect oral cancer. The wavelengths 375 nm and 460 nm, with filters of 479 nm and 525 nm, were designed to excite and detect reduced-form nicotinamide adenine dinucleotide (NADH: 375 nm to 479 nm) and flavin adenine dinucleotide (FAD: 460 nm to 525 nm) autofluorescence. Patients with oral cancer or with precancerous lesions, and a control group with healthy oral mucosae, were enrolled. The lesion in the autofluorescent image was the region of interest. The average intensity and heterogeneity of the NADH and FAD were calculated. Redox ratio (NADH/NADH+FAD) was also computed. A quadratic discriminant analysis (QDA) was used to compute boundaries based on sensitivity and specificity. We analyzed 49 oral cancer lesions, 34 precancerous lesions, and 77 healthy oral mucosae. A boundary (specificity: 0.948; sensitivity: 0.898) between the oral cancer lesions and healthy oral mucosae was validated as redox ratio vs. NADH intensity. Oral cancer and precancerous lesions were also differentiated from healthy oral mucosae (specificity: 0.870; sensitivity: 0.771. redox ratio vs. NADH intensity). The two-channel autofluorescence detection device and analysis of the intensity, heterogeneity, and redox ratio combined with a QDA classifier can be used to differentiate oral cancer and precancerous lesions from healthy oral mucosae.

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Sensing membrane potential changes using de novo designed genetically-encoded voltage indicators
Martin J. Iwanicki, Sohini Mukherjee, Christopher C. Moser, Brian Y. Chow, and Bohdana M. Discher
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA

Developing technologies for optically recording neuronal communication is vital for understanding the basic neural circuitry of the brain. Organic voltage-sensitive dyes have been successful at detecting neural signals at fast temporal resolution (sub-microsecond timescale), yet they cannot be targeted to a specific cell or membrane unless tethered to a protein. Genetically-encoded voltage indicators (GEVIs) can be genetically targeted to cells and membranes, but tend to be dimmer and slower than organic voltage-sensitive dyes. Here, we present our progress on the design and characterization of ultrafast de novo designed genetically-encoded voltage indicators (dnGEVIs) in order to detect changes in membrane potential. These dnGEVIs are based on maquettes, de novo designed 4-α-helical bundle proteins. The voltage-sensing mechanism of these dnGEVIs varies from current GEVIs. Instead of depending on structural rearrangements of the protein for voltage-sensing, dnGEVIs sense voltage with a chain of voltage-sensitive hemes embedded within a transmembrane maquette, where the change in redox states of the hemes modulates the fluorescence of a fluorescent protein attached to the maquette. We have developed a dnGEVI prototype fusion construct by connecting a heme-containing maquette to mOrange2, an orange fluorescent protein. Preliminary results have demonstrated energy transfer between mOrange2 and the maquette during heme reduction. Currently, we are characterizing the energy transfer between a three-heme containing transmembrane maquette and mOrange2. Additionally, we are designing and developing new transmembrane maquettes with different sequences for the purpose of achieving superior detection of changes in neuronal signals.

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Izzetoglu, Meltem

Cerebral edema and oximetry monitoring using NIRS
Meltem Izzetoglu, PhD¹, Shadi Malaeb, MD², Kurtulus Izzetoglu, PhD², Hasan Ayaz, PhD², Banu Onaral, PhD², and Baruch Ben Dor, PhD^2,3
1Villanova University, Villanova, PA; ²Drexel University, Philadelphia, PA; ³InfraScan, Inc., Philadelphia, PA

BACKGROUND: Noninvasive and continuous monitoring of cerebral oxygen saturation and edema development is critical in the timely detection, prevention and treatment of brain injury. Near Infrared Spectroscopy (NIRS) has the capability of monitoring changes in cerebral blood and water content continuously for prolonged time in a safe, noninvasive and portable way.

METHODS: In this pilot study, a NIRS based device is used for the monitoring of cerebral edema and oximetry in a piglet model. Infrascanner, model 3000 is a brain monitoring device based on NIRS technology being developed under US Marines contract as the next generation of the FDA approved hand-held hematoma detector Infrascanner, model 2000 with additional sensors, hardware, software and algorithms to provide cerebral oximetry and edema monitoring capabilities. Initial testing of the prototype is performed on a piglet model with (n=4, HI) and without (n=1, Nx) hypoxia/ischemia to create changes in cerebral oxygen saturation and cause edema development over time.

RESULTS: Piglets with HI showed increased intracranial pressure (ICP) and wet-to-dry brain weight measurement confirmed development of cerebral edema as a result of hypoxic/ischemic insult in comparison to the Nx piglet. In all piglets water signal obtained by the NIRS based brain monitoring device provided good agreement with ICP values and wet-to-dry ratio calculations. Cerebral oximetry measurements in all piglets also showed good correlation with oxygen saturation measurements obtained using external sensors and blood samples.

CONCLUSION: This pilot study on controlled animal model have suggested that with appropriate sensor configurations and analysis algorithms, NIRS technology can effectively monitor changes in cerebral oximetry and edema development noninvasively and continuously. The technology is ready for human testing which will soon start with two approved IRB protocols at the Hospital of University of Pennsylvania, one on controlled hypercapnia conditions in healthy volunteers and the other on traumatic brain injury patients.

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An iterative deconvolution methodology for Cherenkov-excited luminescence imaging by using of irradiation geometry in breast precise external beam radiotherapy
Mengyu Jeremy Jia¹, Petr Bruza¹, Lesley A. Jarvis³, David J. Gladstone^1,2,3, Brian W. Pogue^1,2
1Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755, USA; ²Norris Cotton Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756, USA; ³Department of Medicine, Geisel School of Medicine, Dartmouth College, Hanover, New Hampshire 03755, USA

Cherenkov-excited luminescence scanned imaging (CELSI) is achieved with External Beam Radiotherapy, to map out molecular luminescence intensity or lifetime in tissue. However, images are typically blurred by scattering effects of both Cherenkov and luminescence photons. Motivated by coded-aperture X-ray imaging, we herein proposed an improved modulation-demodulation method aimed at highly resolving those subcutaneous targets in breast tissue, e.g., lymph node. The ‘coded’ illumination could be extracted and customized from the dynamic radiation source in present precise radiation therapy. With CELSI technique, medium region irradiated by this continuously transformed beam can be captured as Cherenkov images, which was then used to iteratively deconvolve the synchronously captured and post-customized luminescence images. The spatial resolution improvements were experimentally demonstrated based on the most commonly used breast treatment plans of Dynamic Wedge (DW), and Intensity Modulated Radiation Therapy (IMRT), respectively. 0.5% Europium solution were filled in 1-mm capillaries as luminescence targets and embedded in a full-size breast phantom with an averaged depth range of 5mm-15mm, which is the typical depth of lymph node. Comparisons based on MTF-curves show that spatial resolution can be improved from 2mm to 0.3mm for a target depth of 10mm within a tissue phantom. It is anticipated that this method can benefit visualization and location for luminescence/fluorescence tagged tumor during radiation therapy.

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Jones, Jake

In vivo, label-free optical biomarkers of age-related differences in wound metabolism
Jake D. Jones, Hallie E. Ramser, Alan E. Woessner, Kyle P. Quinn
Department of Biomedical Engineering, University of Arkansas, Fayetteville, AK

Skin wound healing is often delayed by advanced age, leaving elderly patients at risk for developing chronic wounds. However, it is challenging to discriminate age-related delays from the numerous possible etiologies of wound chronicity. Our lab has recently demonstrated an ability to discriminate delayed healing in diabetic wounds using the autofluorescence of metabolic cofactors, NADH and FAD. Yet, there is still a critical need to understand how these metrics are sensitive to intrinsic aging. The objective of this study is to utilize in vivo label-free multiphoton microscopy (MPM) to characterize differences in wound metabolism between aged (24 months) and young (4 months) wounds in a murine model (n = 23 total mice). Through in vivo MPM, we calculated an optical redox ratio of FAD/(NADH+FAD) autofluorescence and NADH fluorescence lifetime to create maps of cellular metabolism from each wound over 10 days. Temporal changes in the optical redox ratio of the epithelium were observed with a decrease in redox ratio from day 1 to days 3 and 5 (p < 0.0033), followed by an increase in redox ratio by day 10 (p = 0.0111) for both groups. However, aged mice exhibited a higher overall redox ratio (p = 0.0002) than the young mice, and females had a higher redox ratio than males (p = 0.0084). NADH fluorescence lifetime images also revealed temporal changes in the mean lifetime of the epithelium, which correlated (R=0.7793, p<0.0079) with the optical redox ratio. These findings indicate an optical sensitivity to the accumulation of free NADH during keratinocyte proliferation, and that aged skin may have reduced proliferative capacity leading to subsequent delays in healing. These findings suggest that MPM can distinguish age-related delays from the hyperproliferative epidermis of diabetic wounds and may be used to guide specific treatment strategies in the clinic.

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Prediction of return of spontaneous circulation during cardiopulmonary resuscitation using frequency-domain diffuse optical spectroscopy in a pediatric swine model of asphyxial cardiac arrest
Tiffany S. Ko^1,2,3, Constantine D. Mavroudis⁴, Timothy Boorady³, Kobina Mensah-Brown³, Ryan Morgan⁵ Andrew Lautz⁵, George Bratinov⁵, Yuxi Lin⁵, Sejin Jeong⁵, Vinay M Nadkarni⁵, Robert Berg⁵, Robert Sutton⁵ Arjun G. Yodh², Todd J. Kilbaugh⁵, and Daniel J. Licht^3
1Department of Bioengineering and ²Department of Physics and Astronomy, University of Pennsylvania, 210 South 33rd Street, Philadelphia, PA 19104-6321, USA; ³Division of Neurology, ⁵Department of Critical Care and Anesthesia, Children’s Hospital of Philadelphia, 3401 Civic Center Blvd, Philadelphia, PA 19104, USA; ⁴Division of Cardiovascular Surgery, Department of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA

RATIONALE: Pediatric in-hospital arrest results in ~70% in-hospital mortality and significant neurological morbidity among survivors. The development of standardized neuromonitoring tools with demonstrated prognostic value during cardiopulmonary resuscitation (CPR) may enable optimized resuscitation strategies with improved neurological outcomes.

OBJECTIVE: Examine the association between return of spontaneous circulation (ROSC) and cerebral hemodynamics measured non-invasively using frequency-domain diffuse optical spectroscopy (FD-DOS).

METHODS: One-month old swine (n=31) underwent asphyxia for seven minutes followed by ventricular fibrillation to induce cardiac arrest. Resuscitation was conducted for 20 minutes with pauses for cardiac rhythm check every two minutes and eligibility for external defibrillation after 10 minutes.

MEASUREMENTS: Optical measurements of cerebral tissue oxygen saturation (StO₂) and total hemoglobin concentration (THC) were acquired continuously from 10 minutes prior to asphyxiation through the resuscitation period.

MAIN RESULTS: ROSC was achieved in 74.2% of subjects (n=23/31; i.e., survivors). A significant difference in THC and percentage change from baseline (THCp, %) was seen at 5 minutes of CPR between survivors (median [IQR] = 111.0% [103.8, 115.0]) and non-survivors (median [IQR] = 94.7% [90.5, 107.7], p=0.023). At 10 minutes of CPR, both absolute and % change from baseline of StO₂ and THC showed significant differences between survivors and non-survivors.

CONCLUSIONS: Our results demonstrate an early association between non-invasive optical measurements of cerebral hemodynamics and subsequent achievement of ROSC during the conduct of CPR. These data will help define treatment thresholds for real-time optimization of resuscitation strategies and provide a valuable tool to rapidly assess cerebral health in the event of pediatric in-hospital cardiac arrest.

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Kolenc, Olivia

Label-free multiphoton microscopy can characterize metabolic changes in Leigh’s Syndrome
Olivia Kolenc, Ajibola B. Bakare, Isaac Vargas Lopez, Joshua Stabach, Shilpa Iyer, Kyle P. Quinn
Department of Biomedical Engineering, University of Arkansas, Fayetteville, AK

Leigh’s Syndrome (LS), a mitochondrial disease marked by severe psychomotor and developmental impairment in infants and young children, is linked to mitochondrial DNA mutations affecting the electron transport chain (ETC). Development of effective therapies requires understanding the metabolic outcomes of these mutations; however, non-invasive quantitative functional biomarkers for diagnosis and evaluation are lacking. The objective of this study is to evaluate the sensitivity of label-free multiphoton microscopy to functional changes in patient-derived cells with mutations in different ETC protein complexes. Five patient-derived fibroblast cell lines carrying either Complex I (CI) or Complex V (CV) mutations, as well as a normal fibroblast line, were cultured. Using label-free multiphoton microscopy, random fields were imaged using a 20x objective (1.0 NA) (n=4 dishes/cell line). NADH and FAD autofluorescence intensities were isolated and normalized as in previous studies. An optical redox ratio of FAD/(NADH+FAD) was computed from each image field. Mitochondrial organization was also assessed using custom-written software to characterize fractal dimension from NADH intensity and Mitotracker images. A higher optical redox ratio was associated with both CV mutations (p=0.0001) and CI mutations (p=0.0157) compared to normal fibroblasts. While cell lines carrying CV mutations had a higher redox ratio overall, there was no significant difference between redox ratios of cells with CI and CV mutations. These findings suggest that LS cells exhibit increased oxidative phosphorylation activity relative to glucose catabolism. In parallel studies, these cells also had a higher oxygen consumption rate, which may be an effort to compensate for ETC defects. Interestingly, the fractal dimension of mitochondria is significantly higher in cells with CV mutations relative to CI (p=0.0012). Our findings demonstrate the potential of non-invasive, label-free imaging for characterizing LS patient-derived cells and suggest measurements of mitochondrial structure and function to enable discrimination between different mitochondrial DNA mutations.

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Early pressure injury prediction using noninvasive optical methods
Alec Lafontant¹, Michael Neidrauer¹, Michael Weingarten², Rose Ann DiMaria-Ghalili³, Guy Fried⁴, Peter Lewin¹, Leonid Zubkov^1
1Drexel University, School of Biomedical Engineering, Philadelphia, PA 19104, USA; ²Drexel University College of Medicine, Department of Surgery, Philadelphia, PA 19104, USA; ³Drexel University College of Nursing and Health Professions, Philadelphia, PA, USA 19104, USA; ⁴Magee Rehabilitation Hospital, Philadelphia, PA, USA 19102, USA

Pressure injuries (PIs) are a serious, secondary complication of spinal cord injury (SCI) that have the potential to interfere with physical, psychological, and social well-being and to impact overall quality of life. Patients with limited mobility who are unable to off-load their body weight from pressure points over extended periods of time are at a high risk of PI development, most commonly in the sacrococcygeal area. Seventeen rehabilitation patients with spinal cord injuries and nonblanchable erythema were recruited to be monitored using diffuse correlation spectroscopy (DCS) during a two-week period. A measurement protocol was developed that consisted of three different stages in order to predict whether patients’ redness would disappear (PNOs) or develop into an advanced ulcer (POs). During the baseline stage the patient was turned onto his or her side and the probe was held against the area of redness for 1-2 minutes. When lying in the lateral position, POs had blood flow 3-7 times faster than PNOs. The patient then laid supine on top of the probe in order to apply pressure from body weight to the sacrum for 8-10 minutes, where blood flow in POs decreased by 2-4 times more than that in PNOs. Finally, the patient turned back to the lateral position to measure the reperfusion response for 2-3 minutes. During this stage, blood flow in PNOs increased immediately after the release of pressure and returned to baseline values; however in POs, blood flow during reperfusion typically decreased from session to session suggesting degradation of the capillary network which may precede pressure injury advancement. Among the seventeen patients recruited, there were 13 PNOs and 4 POs, all of which were correctly predicted using the data collected from the DCS system.

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Lal Meena, Bharat

Concentration of FAD as a marker for cervical pre-cancer detection
Bharat Lal Meena^1,2, Kiran Pandey⁴ Asha Agarwal⁵, Chayanika Pantola⁶ and Asima Pradhan^1,3
1Department of Physics, Indian Institute of Technology Kanpur, Kanpur, India; ²Department of Physics, University of Rajasthan, Jaipur, India; ³Center for Lasers and Photonics, Indian Institute of Technology Kanpur, Kanpur, India; ⁴Department of Obstetrics and Gynecology, GSVM Medical College, Kanpur, India; ⁵Department of Pathology, Regency Hospital, Kanpur, India; ⁶Department of Pathology, LPS Institute of Cardiology, Kanpur, India

We report here the ex vivo results of an in-house fabricated portable device based on polarized fluorescence measurements in the clinical environment. This device measures the polarized fluorescence and elastic scattering spectra with 405 nm laser and white light sources respectively. The dominating fluorophore with 405 nm excitation is flavin adenine dinucleotide (FAD) with fluorescence peak around 510 nm. The measured spectra are highly modulated by the interplay of scattering and absorption effects. Due to this, valuable information gets masked. To reduce these effects, intrinsic fluorescence was extracted by normalizing polarized fluorescence spectra with polarized elastic scattering spectra obtained. A number of fluorophores contribute to the fluorescence spectra and need to be decoupled to understand their roles in the progression of caner. Gaussian curve fitting analysis has been utilized to decouple the different bands of contributing fluorophores (FAD and Porphyrin). The change in concentration of FAD during disease progression manifests in the change in ratio of total area and FWHM of its Gaussian profile. Receiver Operating Characteristic (ROC) curve analysis has been used to to discriminate different grades of cervical pre-cancer by using the ratio as input parameter. The sensitivity and specificity for discrimination of normal samples from CIN I are 75% and 54.41% respectively. Further, the normal samples can be discriminated from CIN II samples with 100% and 80.88% sensitivity and specificity respectively and the CIN I from CIN II samples can also be discriminated with 100% sensitivity and 90% specificity respectively. The results show that the change in the concentration of (FAD) can be used as a marker to discriminate the different grades of the cancer and biochemical changes at early stage of the cancer can also be monitored with this technique.

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Lee, Hyunyeol

MRI assessment of CMRO₂ changes during sleep
Hyunyeol Lee¹, Alessandra Caporale¹, Pei-Hsin Wu¹, Michael C Langham¹, John A Detre², Richard J Schwab³, Felix W Wehrli^1
1Department of Radiology, University of Pennsylvania, Philadelphia, PA; ²Department of Neurology, University of Pennsylvania, Philadelphia, PA; ³Sleep Medicine Division, Department of Medicine, University of Pennsylvania, Philadelphia, PA

SYNOPSIS; The purpose of this work was to assess hypothesized changes in whole-brain CMRO₂ during sleep by MRI oximetry and simultaneous CBF measurements. Results suggest that during wakefulness CMRO₂ is stable while following onset of sleep decreases by up to 25%.

INTRODUCTION: During slow-wave-sleep synaptic transmissions are reduced, along with brain glucose utilization¹ and cerebral metabolic rate of oxygen (CMRO₂). CMRO₂ can be quantified via Fick’s principle as CMRO₂=Ca·CBF·(SaO₂-SvO₂); where C_a is the blood oxygen carrying capacity CBF, cerebral blood flow, and SaO₂ and SvO₂ are oxygen saturation levels in arterial and venous blood. The MRI-based OxFlow technique2 in combination with radial encoding3 enables quantification of whole-brain SvO₂ and CBF simultaneously with high temporal resolution, yielding CMRO₂with SaO₂measured from a pulse oximeter. Here, we investigated the feasibility of MRI-based assessment of whole-brain CMRO₂ in humans during wakefulness and sleep.

METHODS: MRI was performed at 3T scanner (Siemens Prisma) using a radial OxFlow sequence3 (TR/TE₁/TE₂=88.2/5.5/13.5 ms, FOV=240² mm², voxel size=1x1x5 mm³, flip-angle=15°, velocity-encoding=76.42 cm/s). To evaluate the method stability, data were continuously collected during 32 minutes of wakefulness in five healthy subjects (age range 29 ̶ 33 years; two females), and coefficient of variation (CV) of the measured parameters were evaluated. Additionally, changes of CMRO₂ during sleep were measured in 4 healthy subjects (ages ranging 31 ̶ 36 years; two females) for approximately 1 hour. Volunteers were asked via visual cueing to press the scanner squeeze ball every 3-5 minutes during pre-determined periods, to provide proof of their wakefulness. Time courses of the measured parameters were derived in each subject from 330 temporal frames. Heart rate was derived from pulse oximeter data.

RESULTS AND DISCUSSION: During wakefulness all physiologic parameters were stable during the time-course (Standard error of mean <1.9 and 1.4 for SvO₂ and CBF, resp., Fig. 1a). However, during sleep CMRO₂ decreased by up to 25% due primarily to increased SvO₂, along with reduced heart rate (Fig. 1b). In contrast, CBF and SaO₂ remained invariant. One limitation of this work the uncertainty on the sleep stage in the absence of polysomnography, which requires EEG monitoring during scanning, currently in progress of being implemented.

Figure 1. Time courses of the physiologic parameters quantified by MRI during wakefulness and sleep. a) Mean and standard error of SaO₂, SvO₂, CBF, CMRO₂, averaged across five subjects during wakefulness for 30 minutes. b) SaO₂, SvO₂, CBF, and CMRO₂ time course in a representative subject during wakefulness and sleep. Gray areas represent the period of conscious response after visual cueing, proving wakefulness. CMRO₂ decreases in correspondence to the transition from awake to asleep state (around 10 mins), here by 23%, paralleled by decrease in heart rate (HR) by roughly 14%.

REFERENCES
1. Boyle PJ, Scott JC, Krentz AJ, Nagy RJ, Comstock E, Hoffman C. Diminished brain glucose metabolism is a significant determinant for falling rates of systemic glucose utilization during sleep in normal humans. J Clin Invest. (1994) 93:529-535.
2. Jain V, Langham MC, Wehrli FW. MRI estimation of global brain oxygen consumption rate. J Cereb Blood Flow Metab. (2010) 30:1598-1607.
3. Cao W, Chang YV, Englund EK, Song HK, Barhoum S, Rodgers ZB, Langham MC, Wehrli FW. High-speed whole-brain oximetry by golden-angle radial MRI. Magn Reson Med (2018) 79:217-223.

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Liang, Wenxuan

Nonlinear endomicroscopy for label-free functional histological imaging in vivo
Wenxuan Liang¹, Defu Chen¹, Gunnsteinn Hall¹, Hyeon-Cheol Park¹, Honghua Guan¹, Kristine Glunde², Israel Gannot³, Katherine Luby-Phelps⁴, Mala Mahendroo⁵, Ming-Jun Li⁶, Xingde Li^1
1Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21205 USA; ²The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 USA; ³Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel; ⁴Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; ⁵Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390; ⁶Science and Technology Division, Corning Incorporated, Corning, New York 14831, USA

Fiber-optic nonlinear endomicroscopy holds exciting promise for enabling label-free, functional histological imaging of biological tissues, in vivo, in situ and in real time at histological resolution without the need for tissue removal or processing. However, to detect the weak intrinsic two-photon signal demands high imaging signal-to-noise ratio (SNR), a long-standing challenge faced by previous fiber-optic nonlinear endomicroscopes. Herein we present a recently developed fiber-optic two-photon endomicroscope featuring unprecedented detection sensitivity as well as excellent compactness (~2 mm in diameter). Through myriad systematic innovations, including customizing a pure-silica-core double-clad fiber and a super-achromatic miniature objective (among many others), the overall imaging SNR of our endomicroscope is enhanced by approximately 20-to 50-fold compared with the prior art. Such improved imaging performance not only permits label-free visualization of biological tissues in situ at histological details, but also enables for the first time two-photon metabolic imaging on an endoscopic setting, thereby opening the door to a plethora of translational applications in vivo. For example, by second-harmonic generation (SHG) imaging of fibrillar collagen, the endomicroscope can visualize cervical collagen architecture and its progressive remodeling over the course of pregnancy; further, the abnormal collagen ripening in RU486/mifepristone-induced preterm birth (PTB) mouse models can be distinguished from normal pregnant mice, demonstrating the potential of the endomicroscope for non¬invasive assessment of PTB risk. As another example, via optical redox ratio (i.e. intensity ratio between NADH and FAD) and NADH lifetime imaging of a functional mouse kidney, the dynamic physiological response of free and protein-bound NADH over the ischemia-reperfusion procedure can be interrogated in vivo and in real time at histopathological resolution. Such integration of subcellular structural and functional information is invaluable for evaluating metabolic perturbations (e.g. ischemia-reperfusion injury) to internal organs in pharmacodynamics studies and transplantation surgeries, and for delineating resection margin during tumor surgeries, etc.

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Lin, Yung-Chi

Evaluation of optical properties in breast cancer tissue by NIRS using diffusion fitting method
Yung-Chi Lin¹, Lili Cheng³, Shoko Nioka⁴, Sheng-Hao Tseng², and Pau-Choo Chung^1
1Institute of Computer and Communication Engineering, ²Department of Photonics, and ³Radiology, National Cheng Kung University, Tainan, Taiwan; ⁴Department of Radiology, Medical School of University of Pennsylvania, Philadelphia, PA, USA

There have been advances in investigation of biological tissues using non-invasive optical methods. Among them, diffuse optics can determine absorption and scattering properties of tissues which can be further translated into tissue physiological functions. Although diffuse optics employing spatially resolved method has been demonstrated by many groups to be useful for diagnostic purposes, one of its variants Multi-Wavelength (MW) continuous-wave spectroscopy (CWS) method has not been tested for its effectiveness in evaluation of tissue functions in clinical settings. In this article, we fit the measurement data acquired from a patient-friendly, seven wavelengths NIR device to the photon diffusion theory to obtain optical properties. We had verified that the proposed method was able to effectively determine sample optical properties through a series of tissue simulating phantom studies. This carefully validated MW CWS system was then applied to measure patients with breast cancer for collecting clinical data. Our optical measurement results showed high degree of correlation to clinicians’ biopsy evaluations. This finding suggested that the MW CWS system could be applied for aiding the cancer diagnoses and the method could be a beneficial part to consider with NIR technology.

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Lin, Zhenqu

Redox-associated gene expression in alveolar macrophages after ozone treatment
Zhenwu Lin¹*, He N Xu¹*, Nithyananda Thorenoor², Lin Z Li¹, Lijun Zhang³, Xuesheng Zhang², Yunhua Wang², Xiaojie Liao², and Joanna Floros^2,4
1Department of Radiology, Perelman School of Medicine, University of Pennsylvania; ²Department of Pediatrics, ³Institute of Personized Medicine, and ⁴Department of Obstetrics and Gynecology, The Pennsylvania State University College of Medicine
*Equal contribution to the work

Macrophages play important pro- or anti-inflammatory roles and modulate immune responses. Surfactant protein (SP-A) not only regulates inflammation, but also involves in lung host defense and innate immunity. Our previous studies have shown a decreased survival in Knockout (SP-A-KO) vs wild-type animals with bacterial infection and after oxidative stress. Ozone exposure reduces the ability of mice to survive and impairs the phagocytic ability of alveolar macrophages.
In our current study, we investigated the redox alteration and gene expression profile shift in macrophages from mouse bronchoalveolar lavage four hours after ozone exposure (2 ppm for 3 h). We studied humanized transgenic (hTG) mice carrying the human SP-A2 variant 1A0 and SP-A-KO mice. In each group both males and females were included. The redox state of macrophages was determined using an optical redox imaging technique that measures the fluorescence of reduced nicotinamide adenine dinucleotide NADH and the oxidized flavoproteins Fp in cultured macrophages. The gene expression profile was analyzed by RNA sequencing and bioinformatics analysis. We found that 1) the SP-A2 gene plays a protective role by reducing Fp level (p=0.03) in male (but not female) mice compared to KO mice. Of the differentially expressed genes between male SP-A2 and SP-A-KO mice, 32 genes were identified with p=5.00E-05 – 0.00015, q=0.015-0.0362; 2) the redox state is sex-dependent after ozone-induced oxidative stress, with females exhibiting high oxidation (larger Fp/(NADH+Fp) value) in the presence of SP-A2 than males (p=0.008). Of the differentially expressed genes between male and female mice, 173 genes were identified (p=5.00E-05 – 0.00085, q=0.0060-0.0495). In the absence of SP-A2 (i.e. KO mice) no sex differences were observed.
The human SP-A2 gene plays a protective role against oxidative stress in hTG male mice, with sex differences being observed in redox shift under oxidative stress in the presence of SP-A2.

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Little, Reginald

On the isotopic, nuclear magnetic origin and theory for curing cancer
Reginald B. Little
Stillman College Tuscaloosa, Alabama

Within tumors nanosolutions of H₂¹⁷O, H₂¹⁶O,¹⁵NH₃, and ¹⁴NH³ are proposed to fractionate ¹⁶O and ¹⁷ in H₂O and ¹²C/¹³C, ¹⁴N/¹⁵N, ¹⁶O/¹⁷O, ²⁴Mg/²⁵Mg and ³²S/³³S for enriching tumor with nonprimordial isotopes of ¹³C , ¹⁷O, ²⁵Mg and ³³S relative to primordials of ¹²C, ¹⁴N, ¹⁶O ²⁴Mg, and ³²S for causing cancer. The environmental pollution by nonionizing electromagnetic radiation and low intensity static magnetic fields are proposed to accelerate such fractionation of isotopes in normal cells to accumulate nonprimordial to transform cell to cancer cells and the group of cells for forming tumors. In this work the replacement of primordial isotopes of ¹²C, ¹⁴N, ¹⁶O, ²⁵Mg, ³¹P and ³²S by nonprimordial isotopes of ¹³C, ¹⁵N, ¹⁷O, ²⁵Mg, ³⁰P, ³²P and ³³S and consequent altered nuclear magnetic moments are proposed to alter genes and demonstrated to accelerate the glycolytic process and suppress the Kreb cycle for transforming normal cells to cancerous cells as by causing the Warburg Effect. On the basis of this universal cause of cancer, a theoretical cure is formulated on the basis of selectively listening to these nonprimordial isotopes in the enzymes of glycolysis and Kreb cycle via NV centers in nanodiamond and using the output to frequency modulate many suitable intense radio frequency waves to selectively rotate these nonprimordial isotopes in their enzymes for altering the enzymatic to suppress the glycolysis for selectively killing cancer cells with no effect on normal cells. Simultaneously synchrotron near edge X-ray or UV visible absorptions of these nonprimordial isotopes can selectively demagnetize the enzymes containing them for selectively killing cancer cells. Furthermore, in conjunction with the X-rays, UV rays and RF fields, polarized neutrons may be used to selectively absorb into the rotating nonprimoridal isotopes to transmute ¹³C to ¹⁴N to selectively inactivate enzymes of accelerated glycolysis only in cancer cells.

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Optical imaging reveals metabolic shifts in breast tumor dormancy and recurrence
Megan C. Madonna
Department of Biomedical Engineering, Duke University, Durham, NC

Over 3 million women in the U.S. who have had breast cancer are at risk for recurrence. Dormant cells can rewire their metabolism to survive hostile environments for years before recurring. To capture these key shifts, we have developed a platform to image the metabolic reprogramming of residual disease in a HER2 model of tumor dormancy. This system uses optical imaging of two previously characterized fluorescent probes: 2-NBDG and TMRE to measure glucose uptake (glycolysis) and mitochondrial membrane potential (mitochondrial metabolism), respectively. In this model, tumors are grown in genetically engineered mouse models and mammospheres that exhibit key features of dormancy and recurrence. Doxycycline (Dox) administration induces expression of HER2 leading to mammary tumor formation while withdrawal of Dox induces oncogene down-regulation. Acute and long-term Dox withdrawal model tumor regression and dormancy, respectively. Residual cells can re-initiate proliferation to form a recurrent tumor.

To validate these probes’ collective ability to distinguish proliferation, regression, dormancy, and recurrence, mammospheres were used initially. High 2-NBDG uptake (p<0.05) was characteristic of proliferative cells while regressing cells showed high TMRE uptake (p<0.05). Dormant cells showed moderate uptake of both 2-NBDG and TMRE with decreased 2-NBDG (p<0.05) and increased TMRE (p<0.05) compared to baseline. Finally, Dox was re-introduced to residual cells to image cells’ transition from dormancy to recurrence. Interestingly, 2-NBDG and TMRE uptake both decreased (p<0.05), but ATP output and growth rate was not significantly different from baseline, pointing to a possible change in substrate.

These results validate 2-NBDG and TMRE’s collective ability to delineate each stage’s metabolism and pave the way for similar studies in vivo using a mammary window chamber with GEM-derived xenografts. From here, altered pathways key to survival can be identified, and therapies aimed at killing residual cells entering dormancy or preventing reactivation into recurrence can be developed.

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Mukherjee, Sohini

Designing transmembrane redox active proteins for action potential sensing
Sohini Mukherjee, Martin Iwanicki, Christopher Moser, Bohdana Discher
University of Pennsylvania, Department of Biochemistry and Biophysics, Philadelphia, PA

Our complex behavior is encoded by action potentials, which are changes in electrical potential along the membrane of the nerve cells driven by differences in ion concentrations. To measure these potentials, many voltage-sensitive indicators have been already developed. However, their in vivo applications have not been fully realized due to various combinations of insensitivity, slow kinetics, heavy capacitative loading, phototoxicity, or lack of genetic targetability. The main goal of this work is to design highly improved optical voltage sensors based on genetically encoded de novo designed synthetic redox proteins. These proteins, called maquettes (Maq), are being developed to bind a chain of redox cofactors to form an intra-protein electron transfer pathway. Electric field changes across the membrane will lead to a change in the oxidation state of the maquette cofactor positioned adjacent to a fluorescent protein (FP). We are currently testing the sensitivity of fluorescent proteins to report the oxidation state of a very commonly used cofactor, heme. Our sensor design involves fusing Redox-Sensitive Red Fluorescent Protein, rxRFP, to the voltage sensitive Maq to initiate fast electron transfer (ET) between the Maq and rxRFP that will cause a change in the rxRFP fluorescence signal. The second design involves fusing the FP (mOrange2) with the Maq. Due to the ideal spectral overlap of the fluorescence of mOrange2 with the absorption spectra of the reduced heme, we expect a good Förster resonance energy transfer (FRET) only for reduced heme and not for oxidized heme, resulting in quenching of the fluorescence signal. To optimize the kinetics, sensitivity, and brightness of our genetically targetable sensors, we are working first with water-soluble Maq-FP constructs and measuring the fluorescence quenching dependence on the oxidation state of the heme in aqueous solutions. The successful design principles will be integrated into a transmembrane construct AM-1, which contains chain of 3 hemes. The properties of AM-1 will be tested in vesicles and mammalian cells. These genetically encoded sensors fused to fluorescent proteins are expected to achieve a speed comparable to the best organic voltage sensitive dyes.

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Blood flow-informed light delivery system for photodynamic therapy
Yi Hong Ong^1,2, Joann Miller¹, Malavika Chandra², Timothy C. Zhu¹, Arjun G. Yodh², and Theresa M. Busch^1
1Department of Radiation Oncology, Hospital of the University of Pennsylvania, USA; ²Department of Physics and Astronomy, University of Pennsylvania, USA

The efficacy of photodynamic therapy (PDT) depends on the light delivery, accumulation of photosensitizer and oxygen availability in a tumor. Tissue hypoxia may develop during PDT due to rapid oxygen consumption and PDT-induced vasoconstriction. Rapid decreases in blood flow during light delivery were found to correlate with poor PDT outcome. This relationship has been suggested to be a consequence of ischemia-introduced hypoxia during light delivery that limits the development of damage-creating reactive oxygen species. Vascular response to PDT is therefore an important component of the treatment effect. We hypothesized that blood flow response during PDT can be used in real time to inform the choice of light delivery parameters to conserve tissue perfusion for improved treatment efficacy. A real-time, blood-flow-informed light delivery system was built and tested. Diffuse correlation spectroscopy (DCS) was used to monitor blood flow continuously in radiation-induced fibrosarcoma murine tumors during Photofrin-mediated PDT. PDT treatment begins at high treatment light fluence rate of 150 mWcm^-2. This interactive system measures real time changes in blood flow and automatically attenuates illumination fluence rate to 25 mWcm^-2 when flow reductions exceed a pre-determined threshold (-10%rBF/min) to conserve blood flow. Illumination fluence rate is adjusted back to 150 mWcm^-2 when blood flow recovers to pre-PDT value to shorten treatment length. Our results show that blood-flow-informed light delivery conserves tumor perfusion and improves PDT efficacy as compared to PDT treatment schemes that employed a constant high or low light illumination fluence rate. Long-term therapeutic efficacy was achieved in 40% of mice treated with blood-flow informed PDT as documented by the absence of tumor recurrence within 90 days of PDT. The results suggest that the ability to measure and modulate tumor physiologic properties can provide a means for personalized delivery of PDT dose and improve treatment outcome.

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Orukari, Inema

Optical imaging to assess the effects of glioma growth on brain connectivity
Inema Orukari¹, Joshua S. Siegel², Nicole M. Warrington³, Grant A. Baxter⁴, Adam Q. Bauer⁴, Joshua S. Shimony⁴, Joshua B. Rubin^3,5, and Joseph P. Culver^1,4,6
1Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130; ²Department of Neurology, Washington University in St. Louis, St. Louis, MO 63130; ³Department of Pediatrics, Washington University in St. Louis, St Louis, MO 63130; ⁴Mallinckrodt Institute of Radiology, Washington University in St. Louis, St Louis, MO 63130; ⁵Department of Neuroscience, Washington University in St. Louis, St Louis, MO 63130; ⁶Department of Physics, Washington University in St. Louis, St. Louis, MO 63130

As surgical and therapeutic techniques have improved, glioma patients live longer after therapy with one of the most frequent areas of morbidity being cognitive deficits. Functional connectivity (fc) disruptions are thought to contribute to these deficits. Fc is a measure of the degree of correlation between brain activity in different brain regions. Unfortunately, investigating the effects of glioma growth on fc in humans is complicated by heterogeneity in lesion size, type, and location across subjects. In this study, we evaluated the effects of tumor growth on fc over time in a controlled mouse model of glioma. We show that decrements in fc were found to significantly anti-correlate with increases in tumor burden measurements. Additionally, disruptions in homotopic fc were found in both proximal and distal regions from the tumor. Finally, we explored changes in hemodynamic parameter as a cause of fc disruptions. We found that local hemodynamic changes, including increased hemodynamic lags, may explain the local changes seen in fc, but not the distal changes in fc. We demonstrate that the hemodynamic lag findings in our mouse model are analogous to the finding that human gliomas exhibit hemodynamic lags in tumoral and peri-tumoral tissues. With the establishment of this mouse model, we can now study how different tumor characteristics contribute to fc disruptions in the setting of glioma, which may lead to better patient risk stratification and improved cognitive outcomes for human glioma patients.

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Osharovich, Sofya

Targeted NIR imaging of choline kinase α-overexpressing lung cancer in mice
Sofya Osharovich^1,2, Anatoliy Popov¹, Sunil Singhal³, David Holt⁴, Jim Delikatny^1,2
1Department of Radiology, ²Pharmacology Graduate Group, and ³Department of Surgery, Perelman School of Medicine; ⁴School of Veterinary Medicine, University of Pennsylvania

INTRODUCTION: Choline kinase α (ChoKα) is a lipid kinase that catalyzes the phosphorylation of choline in the Kennedy pathway of phospholipid biosynthesis. ChoKα has been shown to be upregulated in many human cancers, including lung, breast, and prostate, and is associated with an aggressive phenotype, high histological tumor grade, and poor clinical outcome. Our lab has designed and synthesized a series of novel near-infrared fluorescent ChoKα inhibitors that accumulate in tumors in proportion to enzyme expression. Our lead compound, JAS239 (λ_ex = 745 nm, λ_em = 775 nm) has higher quantum yield and lower serum binding compared to the FDA-approved non-specific fluorophore, ICG.

HYPOTHESIS: Our hypothesis is that JAS239 can identify ChoKα-overexpressing lung tumors during fluorescence imaging.

METHODS: Murine lung cancer cells were screened by Western blotting to measure relative ChoKα expression. Naïve and KLN-205 tumor-bearing mice were injected intravenously with 1 mg/kg JAS239 (1% ethanol in saline). Tumors and/or organs were excised and imaged using the IVIS Spectrum (excitation: 730-760 nm, emission: 810-830 nm). Fluorescence was quantified using Living Image software.

RESULTS: The cell line KLN-205 had the highest levels of ChoKα and were used as our ChoKα-overexpression model. Injection of JAS239 into mice with subcutaneous KLN-205 flank tumors led to tumor fluorescence at 4 hours with a tumor:muscle ratio of ~2:1. Preliminary studies showed that JAS239 was able to illuminate small lung metastases in mice bearing subcutaneous KLN-205 flank tumors compared to naive mice, which had no fluorescent puncta.

DISCUSSION/CONCLUSIONS: These studies indicate that JAS239 is a useful tool for identification of ChoKα-overexpressing primary tumors, as well as micrometastases in an intraoperative setting in mice and that this probe could have the potential for translation into the veterinary clinic for tumor margin identification in canine companion animals with spontaneous NSCLC.

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Placental metabolic rate of O₂
Ana E Rodríguez-Soto¹, Michael C Langham¹, Eileen Hwuang² Walter R Witschey¹, Nadav Schwartz³, Pei-Hsin Wu¹, and Felix W Wehrl^1
1Department of Radiology, University of Pennsylvania, Philadelphia, PA; ²Department of Bioengineering, Division of Maternal-Fetal Medicine, University of Pennsylvania, Philadelphia, PA; ³Department of Obstetrics and Gynaecology, Division of Maternal-Fetal Medicine, University of Pennsylvania, Philadelphia, PA

SYNOPSIS: Current imaging tools are limited in their ability to assess placental dysfunction, a major cause of adverse pregnancy outcomes. Here, we present quantitative MRI methods to estimate placental metabolic rate of O₂(PMRO₂) as a surrogate marker of placental dysfunction.
INTRODUCTION: Placental dysfunction is widely accepted as a major cause of common adverse pregnancy outcomes. The development of non-invasive methods for the evaluation of placental oxygen metabolism would provide new insights into the etiology of placental dysfunction and possibly allow intervention at an early stage of gestation. Here, we incorporated quantitative MRI methods including the noninvasive measurement of oxygen saturation and blood flow rate (BFR), to test the feasibility of estimating placental metabolic rate of O2 (PMRO2) in vivo.
METHODS: To date 25 pregnant women (gestational age, GA=29±3wks; 20 normal pregnancies and 5 diagnosed as intrauterine growth restriction (IUGR)) were scanned at 1.5T (Siemens Avanto, Erlangen, Germany) with two flexible body coils combined with spine coils. According to Fick’s Principle: MRO₂= C_a∙BFR∙(S_aO₂-S_vO₂), where C_a is the O₂ carrying capacity of blood and S_aO₂-S_vO₂ is the arterio-venous difference in oxygen saturation. PMRO₂ can then be calculated by subtracting fetal MRO₂ from uterine MRO₂. Blood flow velocity was estimated using cardiac gated 4D and ungated 2D PC-MRI in uterine arteries and umbilical vein (UV), respectively. Oxygen saturation at the ovarian vein and the UV were estimated with T₂-based MR oximetry.^1,2 Fetal venous oxygen saturation was measured using susceptometry-based oximetry³ in the fetal abdominal aorta as it feeds the umbilical arteries. Fetal mass was calculated from fetal volume.⁴
RESULTS: The average PMRO₂ in normal (n=13) and IUGR (n=4) pregnancies were 33.8±20.2 and 14.6±8.4 mLO₂/min/kg, respectively. At this stage of the ongoing study the large difference in mean PMRO₂ did not quite reach statistical significance (p=0.08).
DISCUSSION:The estimated in vivo PMRO₂ are in agreement with those previously reported at late 3rd trimester, despite differences in methods.^5,6 Of note, PMRO₂ has not been previously reported at GA<34 weeks. The data suggest the potential utility of the proposed approach to evaluate complicated pregnancies, the subject of future work.

REFERENCES:
1. Maleki N, Dai W, Alsop DC. (2012) Optimization of background suppression for arterial spin labeling perfusion imaging. MAGMA 25(2):127-33.
2. Rodríguez-Soto AE, Abdulmalik O, Langham MC, Schwartz N, Lee H, Wehrli FW. (2017) T2-prepared balanced steady-state free precession (bSSFP) for quantifying whole-blood oxygen saturation at 1.5T. Magn Reson Med.
3. Fernandez-Seara MA, Techawiboonwong A, Detre JA, Wehrli FW.  (2006) MR susceptometry for measuring global brain oxygen extraction. Magn Reson Med. 55:967-73.
4. Baker PN, Johnson IR, Gowland PA, et al. (1994) Fetal weight estimation by echo-planar magnetic resonance imaging. Lancet 343(8898):644-645.
5. Saini BS, Zhu M, Portnoy S, Porayette P, Lim J, Duan A, Sled JG, Wald R, Windrim R, Macgowan C, Kingdom JC, See M. (2016) OP29.07: Non-invasive in utero measurements of placental oxygen transport using MRI. Ultrasound Obstet Gynecol. 48(S1):148.
6. Richardson B, Nodwell A, Webster K, Alshimmiri M, Gagnon R, Natale R. (1998) Fetal oxygen saturation and fractional extraction at birth and the relationship to measures of acidosis. Am J Obstet Gynecol 178(3):572-579.
ACKNOWLEDGEMENTS: Human Placental Project U01 HD087180. NIH UL1TR001878.

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Handheld oxygenation imaging toward DFU self-screening
Guennadi Saiko, PhD
Oxilight inc, Toronto, Canada

INTRODUCTION: Early recognition of impaired healing is an essential step in the cost-effective management of non-healing wounds. There are no early indicators that can predict successful wound healing. Tissue oxygenation is recognized as critical to successful wound healing. However, at present, only surrogate measures of wound oxygenation are available clinically and include arterial duplex studies, dye-based imaging modalities, laser Doppler and transcutaneous oximetry. Many of these technologies are not available within the confines of the wound healing clinic, and thus access to these studies becomes a barrier in and of itself. Direct measurement of wound oxygenation and perfusion (total hemoglobin) with a handheld device would be a valuable adjunct in the management of complicated chronic wounds. The multispectral imaging technology has the potential to provide real-time clinical information regarding the baseline oxygenation of the wound bed and surrounding tissues. However, the existing oxygenation imagers are too expensive for routine low extremity screening and thus have not received the wide adoption yet. To get the technology widely accepted, a simple, low-cost solution has to be developed. The scope of the present work is to develop a prototype of such solution.
RESULTS: We have developed a small device, which in combination with a smartphone visualizes tissue oxygen saturation. The device acts as a multispectral flash, which illuminates the tissue with a sequence of flashes, each at a predetermined wavelength. The smartphone camera captures the sequence of images (each with illumination at the particular wavelength). The app processes the multispectral image, extract SO2 map and presents it immediately on the smartphone screen.
DISCUSSION The preliminary clinical and usability data shows that the device has the potential to become a standard of care in diabetic foot ulcer screening. The device is simple enough to be used by patients for self-screening.

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Schaefer, Patrick

Fluorescence lifetime Iimaging microscopy of NADH in mitochondrial disorders
Patrick Schaefer¹, Moritz Niederschweiberer², Bjoern von Einem², Angelika Rueck³, Christine A.F. von Arnim², Douglas C. Wallace^1
1Center for Mitochondrial and Epigenomic Medicine at the Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; ²Ulm University, Department of Neurology, Ulm, Germany; ³Ulm University, Core Facility Confocal and Multiphoton Microscopy, Ulm, Germany

Mitochondria play a central role in cellular health by maintaining bioenergetic balance, ion homeostasis as well as regulating cell death. Accordingly, mutations for example in components of the electron transport system result in severe metabolic phenotypes affecting mostly the brain, heart, eye and skeletal muscle¹. Similarly, there is a strong mitochondrial component also in neurodegenerative disorders like Alzheimer’s disease². However, both primary and secondary mitochondrial disorders are characterized by a high heterogeneity in tissues, cell types or mitochondrial subpopulations affected the most, urging the need for reliable markers of mitochondrial function. The NAD/NADH redox state is an important marker as well as mediator of the energetic state of a cell. Performing fluorescence lifetime imaging microscopy (FLIM) of NADH autofluorescence allows distinguishing free (400ps) and protein-bound (1500ps – 4000ps) NADH. This provides information about both cellular redox state as well as cellular energy metabolism³. Here we demonstrate the application on primary hippocampal neurons overexpressing Alzheimer’s disease related proteins. These comprise the amyloid precursor protein (APP), APP harboring a mutation prone to an increased Aβ42/40 ratio (APPswe) and the beta-secretase 1 (BACE1) resulting in more amyloidogenic cleavage and higher Aβ production. Performing innovative parallel NADH FLIM – pH imaging provides a subcellular readout of mitochondrial function in the neurons, allowing to distinguish between somatic and neuritic mitochondria. We could demonstrate, that both overexpression of BACE1 as well as APPswe result in a reduced endogenous respiration primarily in the somatic mitochondria. A second application focuses on primary mitochondrial disorders, using NADH FLIM to decipher the effect of different mitochondrial mutations on the NAD+/NADH redox state with a special focus on how heteroplasmy segregates within the mitochondria of the same cell. In summary, here we demonstrate how NADH FLIM can help unscrambling mitochondrial function on the subcellular level both in primary and secondary mitochondrial disorders.
1. Wallace DC. (2013) A mitochondrial bioenergetic etiology of disease. J Clin Invest 123:1405-1412.
2. Coskun P, Wyrembak J, Schriner SE, Chen HW, Marciniack C, Laferla F, & Wallace DC. (2012) A mitochondrial etiology of Alzheimer and Parkinson disease. Biochim Biophys Acta 1820:553-564.
3. Schaefer PM, Hilpert D, Niederschweiberer M, Neuhauser L, Kalinina S, Calzia E, Rueck A, von Einem B, & von Arnim CAF. (2017) Mitochondrial matrix pH as a decisive factor in neurometabolic imaging. Neurophotonics 4:045004.

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High-resolution mapping of stiffness heterogeneity in pancreatic tumors as it affects drug delivery
Phuong Vincent, Kayla Marra, Jason Gunn, Michael Nieskoski, Kimberley Samkoe and Brian Pogue
Thayer School of Engineering, Dartmouth College, Hanover, NH 03755

The five-year survival rate of patients who suffer from locally advanced pancreatic ductal adenocarcinoma (PDAC) has remained relatively the same over the past 40 years. In addition to novel therapy approaches, it is critical to understand the pathophysiology of tumor microenvironment since pharmacological treatments directly depend on the tumor intrinsic characteristics. Therefore, this project was conducted to validate stiffness heterogeneity induced by collagen as a major drug delivery barrier in PDAC tumors. Two human pancreatic cell lines AsPC-1 and BxPC-3 were assessed due to their stroma difference. A sensitive fiber optic pressure sensor was coupled with a motorized xyz table to map out the spatial distribution of tumor stiffness in mouse model. The tumors were orthotopically implanted and resected when the tumor size had reached 1cm diameter. The imaging sample was prepared by first embedding the tumors into 1.5% agar gel. Then, the gel was sliced in half to provide a level surface for stiffness mapping. Three small pins were inserted into the tumors to provide markers that were used as matching points with histology data. A LabVIEW program was created to detect the surface of the tumor and to maintain a constant strain across the surface. The stiffness map on a scale of 300micron was co-registered with histology data on collagen distribution (Masson’s Trichrome), hyaluronic acid (HABP-1), necrosis area (H&E), and functional vascular network (Lectin). Preliminary results have showed a strong spatial correlation between collagen density and stiffness values. Regional analysis on collagen complexity and hyaluronic acid content will be performed to highlight the relationship with stiffness heterogeneity. The potential correlations between these tumor microenvironment parameters and the physical stiffness of tumor tissues suggest promising therapy targets that could improve the efficiency of drug transport, from which chemotherapy and other pharmacological treatments will greatly benefit from.

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Non-invasively quantification of placenta oxygenation with frequency domain diffuse optical spectroscopy
Lin Wang¹, Tiffany Ko², Lian He¹, Wesley Baker¹, Jeffrey Cochran¹, Kenneth Abramson¹, David Busch¹, Venki Kavuri³, Ashwin Parthasarathy⁴, Leonid Zubkov, Alex Acker⁵, Arjun Yodh¹, Nadav Schwartz^1
1Department of Physics & Astronomy, University of Pennsylvania; ²Department of Bioengineering, University of Pennsylvania; ³Masimo Corporation ⁴Department of Electrical. Engineering, University of South Florida; ⁵Department of OB/GYN, Perelman School of Medicine, University of Pennsylvania

Monitoring placenta oxygenation is critical to ensure a healthy pregnancy outcome. Evaluating placenta oxygenation function to detect fetal growth restriction requires measuring the oxy and deoxy hemoglobin concentration quantitatively. Previously placenta oxygenation was measured by using commercial Continuous-Wave Near Infrared Spectroscopy (CW NIRS). However, their longest Source Detector Separation (SDS) is 4.5 cm, which is too small to provide enough light penetration depth to explore placenta. Also, the CW NIRS measurement does not give access to absolute values of total hemoglobin or oxygen saturation but only their changes. Here we report on a state-of-the-art Frequency Domain Diffuse Optical Spectroscopy (FD-DOS) system with high modulation depth, high signal to noise and large SDS range. Importantly, the optical system is integrated with an ultrasound transducer which provides placenta depth and position and thus helps improve measurement fidelity. The heterodyne FD-DOS instrument employs three different wavelengths lasers that were amplitude-modulated at 100 MHz. Nearly 100% of modulation depth was achieved to improve signal to noise and reduce needed laser power. A reference signal at a slightly different radio frequency, 100.2 MHz, permits heterodyne down-conversion of the signal at lower-frequency, (0.2 MHz). A high sampling rate Lock-In amplifier compares the reference signal and detected signal to obtain both amplitude and phase shift. To characterize the system, we have tested the instrument on tissue simulating phantom (homogeneous) with realistic known optical properties (μ_a=0.09 cm^-1, μ_s'=8.9 cm^-1). With SDS up to 10 cm, the system can detect optical properties with less than 10% error. We have utilized this FD-DOS/Ultrasound system in a preliminary clinical study. Specifically, physiologic measurements of total hemoglobin concentration (HTb, μmol/L) and tissue oxygen saturation (StO2, %) were measured in healthy mothers. We see higher HTb and StO2 in placenta than superficial tissue, which will need to be tested by more measurements.

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Wen, Yu

Optical redox imaging detects the effects of DEK oncogene knock down on the redox state of breast cancer cells
Yu Wen^1,2, He N. Xu², Zhenwu Lin², Lisa Privette Vinnedge³, Lin Z. Li^2,4
1Rutgers Cancer Institute of New Jersey, New Brunswick, NJ; ²Department of Radiology, Perelman School of Medicine, University of Pennsylvania; ³Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH; ⁴Abramson Cancer Center & Institute of Translational Medicine and Therapeutics, Perelman School of Medicine, University of Pennsylvania

Optical redox imaging (ORI), based on collecting the fluorescence of endogenous NADH and oxidized flavoproteins (Fp containing flavin adenine dinucleotide, i.e., FAD), provides sensitive indicators of cellular metabolism. To investigate the potential of ORI indices (such as NADH, FAD, and their ratio) as prognostic biomarkers in breast cancer, we previously reported that higher metastatic potential is associated with larger FAD redox ratio (FAD/(FAD+NADH)) in tumor xenografts and cultured cells. Meanwhile, we noticed that DEK oncogene over-expression reprograms metabolism, enhances cell proliferation, invasion, and metastasis. Therefore in this study we hypothesize that DEK gene activity may influence ORI indices. Using lentiviral shRNA, DEK gene expression was efficiently knocked down in MDA-MB-231 cells. FAD fluorescence and FAD redox ratio in polyclonal cells with DEK knock down was significantly lower than that in control cells. Consistently, when the knockdown of DEK was not sustained in the late passages of polyclonal cell populations, the cancer cells did not show significantly different FAD redox ratio and FAD intensity versus the control cells. This is the first direct proof of the concept that oncogene activities could mediate ORI-detected cellular redox state. Our results support ORI indices as potential biomarkers for the prognosis of breast cancer.

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Williams, Catrin

Biological response to separated microwave electric and magnetic fields
Catrin F. Williams¹, Gilles M. Geroni¹, Antoine Pirog¹, David Lloyd^1,2, Jonathan Lees¹, Nick Clark¹, and Adrian Porch^1
1School of Engineering, Queen’s Buildings, Cardiff University, Cardiff CF24 3AA, Wales, UK; ²School of Biosciences, Cardiff University, Main Building, Cardiff CF10 3AT, Wales, UK

Microwaves are ubiquitous in our modern, urban environment. The thermal effects of these electromagnetic fields on biological systems have been well researched. However, possible non-thermal effects remain a controversial subject. Our work utilizes the bioluminescent marine bacterium, Vibrio fischeri, as a novel biosensor to probe the effects of low power, pulsed microwave (2.45 GHz) magnetic and electric fields. We observed differential responses in light output from V. fischeri, when this bacterium was exposed to separated electric and magnetic fields in a TM₀₁₀ mode resonant cavity. Exposure to electric field (0.3 W rms, 6 kV/m) resulted in fast responses with an increase in light emission in excess of 200%, followed by decline and sharp recovery when radiation was stopped. Magnetic field exposure (32 W rms, 183 A/m) elicited no measurable responses, even at 100-fold higher available power. We suggest that the observed electric field effects are only partially the results of small, but rapid, heating effects (using Mathcad modelling and thermal imaging). However, we cannot exclude the possible non-thermal mechanisms contributing to these stimulatory and inhibitory effects on the biological processes involved in photon emission from V. fischeri. The ultimate goal of this project is to microscopically image these biological effects in real-time using custom made luminophores, to elucidate the mode of action of microwaves at the molecular level.

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Wu, Melissa

Model impact on calibrating extracerebral contributions to DCS CBF measurements
Melissa M Wu¹, Parisa Farzam¹, Parya Farzam¹, Jason Z. Qu², Juliette Selb³, Maria A. Franceschini¹, Stefan A. Carp^1
1Athinoula A. Martinos Center, Massachusetts General Hospital, Charlestown, MA 02129; ²Dept. of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, MA 02114 ³Montreal Heart Institute, Montreal, QC H1T 1C8

Diffuse correlation spectroscopy (DCS) is increasingly being used as a non-invasive method to measure tissue perfusion. However, extending the use of this technique to adult brain monitoring requires addressing the influence of extra cerebral contributions on DCS measurements. For reflectance measurements, light has traveled mostly through extracerebral tissues, resulting in potentially skewed cerebral blood flow (CBF) estimates due to systemic physiology variations. Recently, a method to calibrate the influence of extracerebral contributions has been proposed by Baker et al., where, after placing the DCS fiber probe on the head, pressure is briefly applied under continuous monitoring. This is expected to cause a significant reduction in skin blood flow without having any influence on cerebral perfusion. If properly adjusted, a multi-layer light transport model should recover superficial blood flow changes with minimal perturbations to CBF values. In this investigation, we report the performance of two Monte Carlo based models in conjunction with a pressure modulation maneuver on a healthy volunteer and on cardiac surgery patients. The first model assumes a slab, semi-infinite layer geometry, while the second uses a realistic head-shaped layered structure derived from the external surface of a 3D MRI scan. Both models are used to create adjustable 2 (scalp & skull vs. brain) and 3 layer (scalp, skull, and brain) geometries. The 3 layer model based on the realistic geometry appears to provide more repeatable and physiologically meaningful results.

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Wu, Pei-Hsin

MRI evaluation of cerebrovascular reactivity in obstructive sleep apnea
Pei-Hsin Wu¹, Ana E Rodríguez-Soto¹, Michael C Langham¹, John A Detre², Richard J Schwab³, Andrew Wiemken³, Alessandra Caporale¹, and Felix W Wehrli^{1 1}Department of Radiology, University of Pennsylvania, Philadelphia, PA, United States; ²Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA, United States; ³Division of Sleep Medicine, Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States

OBJECTIVE: Patients with obstructive sleep apnea (OSA) are at elevated risk for developing cardiovascular disease and stroke. Here we evaluated the hypothesis that OSA entails altered cerebrovascular reactivity (CVR). Toward this goal, we measured the breath-hold index (BHI), defined as the temporal changes in global cerebral blood flow (CBF) during successive breath-holds in subjects with and without OSA at 3T field strength.

METHODS: A 10-min paradigm consisting of five breath-hold (BH) cycles, each lasting 24s followed by 66s breathing recovery, was applied. Blood flow velocity data was collected at a temporal resolution of 1.3s. Blood flow rate in the superior sagittal sinus (SSS) was determined via phase-contrast MRI, and upscaled to total CBF (tCBF) by using the information from a calibration scan where velocity was measured at baseline in the internal carotid and vertebral arteries. Total CBF was subsequently normalized to 100g of brain tissue. BHI was determined as the rate of change dCBF/dt within a BH period and averaged over 5 cycles (Fig. 1). Twenty-eight subjects with OSA (mean age 50.0 years, BMI 31.0, and apnea–hypopnea index [AHI] 41.8) and 22 controls (mean age 49.0 years, BMI 31.2, AHI 3.6) were recruited.

RESULTS: Compared with controls, subjects with OSA had a significantly greater BHI (86.52 versus 70.36 mL/min²/100g, P = 0.03). BHI based on SSS flow velocity was also significantly different between groups (0.52 versus 0.42 cm/s², P = 0.01) (Fig. 2). These surprising results are in overall agreement with a recent study evaluating CVR on the basis of an exogenous hypercapnic stimulus (Ryan et al, J Stroke Cerebrovasc Dis. 2018). Finally, there were no significant differences in either tCBF or blood flow velocity at baseline or peak breath-hold between the two groups. Conclusions: Greater CVR in OSA has been conjectured to arise as a compensatory mechanism; however, results should be interpreted with caution given the limited sample size.

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Wu, Shulian

Quantitative evaluation of redox ratio during breast cancer chemotherapy using two-photon imaging
Shulian Wu^1,2, Qinggong Tang², Zhifang Li¹, Yu Chen², Hui Li^1
1College of Photonic and Electronic Engineering, Fujian Normal University, Fujian Provincial Key Laboratory of Photonic Technology, Key Laboratory of Optoelectronic Science and Technology for Medicine, Ministry of Education, Fuzhou, 350007, China; ²Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA

Preoperative neoadjuvant treatment in locally advanced breast cancer is recognized as effective adjuvant therapy, as it improves treatment outcomes. However, the potential complications remain a threat, so there is an urgent clinical need to assess both the tumor response and changes in its microenvironment using non-invasive and precise identification techniques. Here, two-photon microscopy was employed to detect metabolic rate in breast cancer progression and recession throughout chemotherapy. NADH intensity, FAD intensity, and optical redox ratio obtained from auto-fluorescence signal was calculated to study the effect of chemotherapy. Results indicate that these parameters are potential indicators for evaluating breast tumors and their microenvironment changes during progression and chemotherapy.

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Optogenetic pacing in Drosophila using red-light
Jing Men¹, Jason Jerwick¹, Rudolph Tanzi², Airong Li², Chao Zhou^1,3,4
1Department of Bioengineering, ³Department of Electrical and Computer Engineering, ⁴Center for Photonics and Nanoelectronics, Lehigh University, Bethlehem, PA-18015, USA; ²Genetics and Aging Research Unit, Department of Neurology, Massachusetts General Institute for Neurodegenerative Diseases, Massachusetts General Hospital, Boston, MA-02114, USA

INTRODUCTION: Cardiac optogenetics is an emerging technology that allows the control of cardiac functions with high spatial and temporal precisions, and is a promising alternative to electrical pacing methods in animal models^1-4. In the recent years, optogenetic cardiac control has been largely limited by insufficient optical penetration depth in tissues. Red light excitation has been increasingly studied in optogenetics because tissue absorption and scattering reduce rapidly with wavelengths longer than 600 nm⁵. Drosophila melanogaster is an important model organism for studying heart development and cardiac physiology^3,6,7. Previously, we have successfully demonstrated optogenetic pacing in Drosophila melanogaster using blue light³. In this study, we explore the feasibility of red-light optogenetic pacing in Drosophila models.

MATERIALS AND METHODS: We developed two transgenic Drosophila organisms with excitatory and inhibitory opsins (ReaChR and NpHR respectively) expressed in the heart. We developed an integrated red-light excitation and optical coherence microscopy (OCM) imaging system (Fig. 1a) to demonstrate cardiac control throughout the life cycle of these intact Drosophila specimens in real time.

Figure 1. (a) Integrated red-light stimulation and OCM imaging system; M-mode OCM images demonstrate (b) increase of heart rate (HR) of a ReaChR fly; (c) decrease of HR of a NpHR fly; and (d) inhibiting heartbeat of a NpHR fly for 10 s using red-light.

RESULTS AND DISCUSSION: We expressed ReaChR and NpHR in the heart of Drosophila, and mimicked restorable tachycardia (Fig. 1b), bradycardia (Fig. 1c), and cardiac arrest (Fig. 1d) through non-invasive red-light cardiac control at the different developmental stages. The heartbeat of Drosophila can be precisely manipulated in real time by modifying the frequency and pulse width of the excitation light.

CONCLUSIONS: Red-light excitation of opsins expressed in intact Drosophila was demonstrated as an effective method for pacemaker control. It holds the potential to become a promising method for studying cardiac pathologies or exploring novel pacing therapies for various cardiovascular diseases.

REFERENCES:
1. T. Bruegmann, D. Malan, M. Hesse, T. Beiert, C. J. Fuegemann, B. K. Fleischmann, and P. Sasse (2010) Optogenetic control of heart muscle in vitro and in vivo. Nature Methods 7:897-900.
2. A. B. Arrenberg, D. Y. Stainier, H. Baier, and J. Huisken (2010) Optogenetic control of cardiac function. Science 330:971-974.
3. A. Alex, A. Li, R. E. Tanzi, and C. Zhou (2015) Optogenetic pacing in Drosophila melanogaster. Science Advances 1:e1500639.
4. U. Nussinovitch and L. Gepstein (2015) Optogenetics for in vivo cardiac pacing and resynchronization therapies. Nature Biotechnology 33:750-754.
5. J. Y. Lin, P. M. Knutsen, A. Muller, D. Kleinfeld, and R. Y. Tsien (2013) ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation. Nature Neuroscience 16:1499-1508.
6. J. Men, Y. Huang, J. Solanki, X. Zeng, A. Alex, J. Jerwick, Z. Zhang, R. E. Tanzi, A. Li, and C. Zhou (2016) Optical coherence tomography for brain imaging and developmental biology. IEEE Journal of Selected Topics in Quantum Electronics 22:120-132.
7. J. Men, J. Jerwick, P. Wu, M. Chen, A. Alex, Y. Ma, R. E. Tanzi, A. Li, and C. Zhou (2016) Drosophila preparation and longitudinal imaging of heart function in vivo using Optical Coherence Microscopy (OCM). Journal of Visualized Experiments e55002-e55002.

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Zhou, Lanlan

Photothermal therapy of malignant mesothelioma with delaminated MXene Ti₃C₂
Lanlan Zhou¹, Fayan Meng², Babak Anasori², Yury Gogotsi², and Wafik S. El-Deiry^1
1Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Medical Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA 19111; ²Department of Materials Science and Engineering and A. J. Drexel Nanomaterials Institute, Drexel University, Philadelphia, PA 19104

Malignant mesothelioma is an aggressive and deadly form of cancer that develops in the thin layer of tissue surrounding the majority of internal organs (mesothelium). Malignant mesothelioma has no known cure and has a very poor prognosis because of late diagnosis and limited usefulness of standard treatments. We are exploring newer treatments to tackle this deadly disease. Photothermal therapy is an emerging noninvasive spatiotemporal selective therapeutic strategy that employs near-infrared photoabsorbers to generate heat for thermal ablation of cancer cells. The therapeutic efficacy of photothermal therapy significantly depends on the transformation of light to sufficient heat with photothermal agents. MXene Ti₃C₂ is an effective two-dimensional light-to-heat conversion material. The internal light-to-heat conversion efficiency of MXene Ti₃C₂ was measured to be 100%, indicating a perfect energy conversion. In this study we report for the first time using the MXene Ti₃C₂nanoplatform as a photothermal agent to efficiently ablate malignant mesothelioma. First, we prepared two-dimensional MXene Ti₃C₂ and UV-vis-NIR spectrum of Ti₃C₂ has a strong absorbance at the region of 790 nm with a high light extinction coefficient of 26.081 L g^-1cm^-1 and a good photothermal conversion efficiency of 22%. Photothermal heating curves show temperatures of different concentration of MXene Ti₃C₂ increase steadily with irradiation of 808 nm laser. Recycling heating profiles demonstrate that MXene Ti₃C₂ can be repeatedly heated stably. Next, the effects of MXene Ti₃C₂ on normal and mesothelioma cell lines were tested with CellTiter-Glo® luminescent cell viability assays. MXene Ti₃C₂ has no toxicity to cells at the tested concentration range. Light microscopy revealed that MXene Ti₃C₂ can accumulate inside of mesothelioma cells but not in normal cells. There was no red blood cell hemolysis within the tested concentration range and the hemolysis ratio was 0.5% even at ten times higher than the test concentration. Then, normal and mesothelioma cells were treated with and without MXene Ti₃C₂ for 24~48 hours followed by laser irradiation. CellTiter-Glo® luminescent cell viability assays and colony formation assays validated that MXene Ti₃C₂-treated mesothelioma cells were eliminated by laser irradiation while normal cells and nontreated mesothelioma were spared. Lastly, we established a mesothelioma mouse xenograft model to evaluate the therapeutic effect of MXene Ti₃C₂ on mesothelioma in vivo. We are also pursuing the combination of MXene Ti₃C₂-based photothermal therapy and current therapies for mesothelioma. Our results indicate that MXene Ti₃C₂ is a promising photothermal agent in therapy of mesothelioma.

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Zhu, Caigang

Optical spectroscopy of vascularity and metabolism in flank tumors
Caigang Zhu¹, Hannah L. Martin¹, Brian T. Crouch¹, Amy F. Martinez², Martin Li¹, Gregory M. Palmer³, Mark W. Dewhirst³, and Nimmi Ramanujam^1
1Department of Biomedical Engineering, Duke University, Durham, NC 27708; ²Office of Research, Vanderbilt University Medical Center, Nashville, TN 37232; ³ Department of Radiation Oncology, Duke University, Durham, NC 27710

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. Considering the importance of metabolism and microenvironment to cancer biology, there are surprisingly few techniques available to provide a systems-level approach to measure metabolism and tumor-associated vasculature in vivo. 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 in vivo optical spectroscopy of tissue vascular parameters, glucose uptake, and MMP in a flank tumor model which is most often used for therapeutic studies. Our optical data shows that 4T1 solid tumors have decreased baseline SO2, increased hemoglobin concentration ([Hb]), increased absorption levels, decreased scattering levels, increased glucose uptake, and increased MMP compared to normal flank tissues. The optical measure of vasculature and metabolism shows a strong positive correlation between metabolic endpoints and their corresponding vascular endpoints in 4T1 tumors. Glucose uptake is also positively correlated with MMP in 4T1 tumors. Our study demonstrates the potential of optical spectroscopy as an effective tool to quantify the vascularity and metabolic phenotype of a tumor, towards understanding the mechanisms underlying cancer progression, metastasis, and resistance to therapies.