Publications by authors named "Mary-Ann Mycek"

49 Publications

Reconstruction of optical coefficients in turbid media using time-resolved reflectance and calibration-free instrument response functions.

Biomed Opt Express 2022 Mar 22;13(3):1595-1608. Epub 2022 Feb 22.

Miami University, Department of Physics, Oxford, OH 45056, USA.

Measurements of time-resolved reflectance from a homogenous turbid medium can be employed to retrieve the absolute values of its optical transport coefficients. However, the uncertainty in the temporal shift of the experimentally determined instrument response function (IRF) with respect to the real system response can lead to errors in optical property reconstructions. Instrument noise and measurement of the IRF in a reflectance geometry can exacerbate these errors. Here, we examine three reconstruction approaches that avoid requiring direct measurements of photon launch times. They work by (a) fitting relative shapes of the reflectance profile with a pre-determined constraint on the scattering coefficient, (b) calibrating launch-time differences via a reference sample, and (c) freely fitting for the launch-time difference within the inverse problem. Analysis methods that can place a tight bound on the scattering coefficient can produce errors within 5-15% for both absorption and scattering at source-detector separations of 10 and 15 mm. Including the time-shift in the fitting procedure also recovered optical coefficients to under 20% but showed large crosstalk between extracted scattering and absorption coefficients. We find that the uncertainty in the temporal shift greatly impacts the reconstructed reduced scattering coefficient compared to absorption.
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http://dx.doi.org/10.1364/BOE.447685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8973157PMC
March 2022

Noninvasive Optical Assessment of Implanted Tissue-Engineered Construct Success .

Tissue Eng Part C Methods 2021 05;27(5):287-295

Department of Biomedical Engineering, University of Michigan College of Engineering, Ann Arbor, Michigan, USA.

Quantitative diffuse reflectance spectroscopy (DRS) was developed for label-free, noninvasive, and real-time assessment of implanted tissue-engineered devices manufactured from primary human oral keratinocytes (six batches in two 5-patient cohorts). Constructs were implanted in a murine model for 1 and 3 weeks. DRS evaluated construct success using optical absorption (hemoglobin concentration and oxygenation, attributed to revascularization) and optical scattering (attributed to cellular density and layer thickness). Destructive pre- and postimplantation histology distinguished experimental control from stressed constructs, whereas noninvasive preimplantation measures of keratinocyte glucose consumption and residual glucose in spent culture media did not. In constructs implanted for 1 week, DRS distinguished control due to stressed and compromised from healthy constructs. In constructs implanted for 3 weeks, DRS identified constructs with higher postimplantation success. These results suggest that quantitative DRS is a promising, clinically compatible technology for rapid, noninvasive, and localized tissue assessment to characterize tissue-engineered construct success . Impact statement Despite the recent advance in tissue engineering and regenerative medicine, there is still a lack of nondestructive tools to longitudinally monitor the implanted tissue-engineered devices. In this study, we demonstrate the potential of quantitative diffuse reflectance spectroscopy as a clinically viable technique for noninvasive, label-free, and rapid characterization of graft success .
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http://dx.doi.org/10.1089/ten.TEC.2021.0018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140358PMC
May 2021

Needle-compatible miniaturized optoelectronic sensor for pancreatic cancer detection.

Sci Adv 2020 11 20;6(47). Epub 2020 Nov 20.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.

Pancreatic cancer is one of the deadliest cancers, with a 5-year survival rate of <10%. The current approach to confirming a tissue diagnosis, endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), requires a time-consuming, qualitative cytology analysis and may be limited because of sampling error. We designed and engineered a miniaturized optoelectronic sensor to assist in situ, real-time, and objective evaluation of human pancreatic tissues during EUS-FNA. A proof-of-concept prototype sensor, compatible with a 19-gauge hollow-needle commercially available for EUS-FNA, was constructed using microsized optoelectronic chips and microfabrication techniques to perform multisite tissue optical sensing. In our bench-top verification and pilot validation during surgery on freshly excised human pancreatic tissues (four patients), the fabricated sensors showed a comparable performance to our previous fiber-based system. The flexibility in source-detector configuration using microsized chips potentially allows for various light-based sensing techniques inside a confined channel such as a hollow needle or endoscopy.
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http://dx.doi.org/10.1126/sciadv.abc1746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679167PMC
November 2020

Direct estimation of the reduced scattering coefficient from experimentally measured time-resolved reflectance via Monte Carlo based lookup tables.

Biomed Opt Express 2020 Aug 16;11(8):4366-4378. Epub 2020 Jul 16.

Department of Physics, Miami University, Oxford, OH 45056, USA.

A heuristic method for estimating the reduced scattering coefficient (µ') of turbid media using time-resolved reflectance is presented. The technique requires measurements of the distributions of times-of-flight (DTOF) of photons arriving at two identical detection channels placed at unique distances relative to a source. Measured temporal shifts in DTOF peak intensities at the two channels were used to estimate µ' of the medium using Monte Carlo (MC) simulation-based lookup tables. MC simulations were used to compute temporal shifts in modeled reflectance at experimentally employed source-detector separations (SDS) for media spanning a wide range of optical properties to construct look up tables. Experiments in Intralipid (IL) phantoms demonstrated that we could retrieve µ' with errors ranging between 6-25% of expected (literature) values, using reflectance measured across 650-800 nm and SDS of 5-15 mm. Advantages of the technique include direct processing of measured data without requiring iterative non-linear curve fitting. We also discuss applicability of this approach for media with low scattering coefficients where the commonly employed diffusion theory analysis could be inaccurate, with practical recommendations for use.
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http://dx.doi.org/10.1364/BOE.398256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449726PMC
August 2020

Optical Metric Assessed Engineered Tissues Over a Range of Viability States.

Tissue Eng Part C Methods 2019 05;25(5):305-313

1 Department of Biomedical Engineering, University of Michigan College of Engineering and Medical School, Ann Arbor, Michigan.

Many conventional methods to assess engineered tissue morphology and viability are destructive techniques with limited utility for tissue constructs intended for implantation in patients. Sterile label-free optical molecular imaging methods analyzed tissue endogenous fluorophores without staining, noninvasively and quantitatively assessing engineered tissue, in lieu of destructive assessment methods. The objective of this study is to further investigate label-free optical metrics and their correlation with destructive methods. Tissue-engineered constructs ( = 33 constructs) fabricated with primary human oral keratinocytes ( = 10 patients) under control, thermal stress, and rapamycin treatment manufacturing conditions exhibited a range of tissue viability states, as evaluated by quantitative histology scoring, WST-1 assay, Ki-67 immunostaining imaging, and label-free optical molecular imaging methods. Both histology sections of fixed tissues and cross-sectioned label-free optical images of living tissues provided quantitative spatially selective information on local tissue morphology, but optical methods noninvasively characterized both local tissue morphology and cellular viability at the same living tissue site. Furthermore, optical metrics noninvasively assessed living tissue viability with a statistical significance consistent with the destructive tissue assays WST-1 and histology. Over the range of cell viability states created experimentally, optical metrics noninvasively and quantitatively characterized living tissue viability and correlated with the destructive WST-1 tissue assay. By providing, under sterile conditions, noninvasive metrics that were comparable with conventional destructive tissue assays, label-free optical molecular imaging has the potential to monitor and assess engineered tissue construct viability before surgical implantation.
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http://dx.doi.org/10.1089/ten.TEC.2018.0344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535959PMC
May 2019

Hybrid Monte Carlo simulation with ray tracing for fluorescence measurements in turbid media.

Opt Lett 2018 Aug;43(16):3846-3849

We present a hybrid Monte Carlo simulation method with geometrical ray tracing (hMC-GRT) to model fluorescence excitation and detection in turbid media by optical imaging or spectroscopy systems employing a variety of optical components. hMC-GRT computational verification was achieved via reflectance and fluorescence simulations on epithelial tissue models in comparison with a standard Monte Carlo code. The mean difference between the two simulations was less than 5%. hMC-GRT experimental verification employed depth-sensitive steady-state fluorescence measurements using an aspherical lens on two-layered tissue phantoms. hMC-GRT predictions agreed well with experimental results, achieving less than 3.5% error for measurements at the phantom surface. Verification results demonstrate that the hMC-GRT simulation has the potential to become a useful computational toolbox for designing tissue fluorescence imaging and spectroscopy systems. In addition, the hMC-GRT approach enables a wide variety of applications for computational modeling of fluorescence in turbid media. The source codes are available at https://github.com/ubioptronics/hMC-GRT.
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http://dx.doi.org/10.1364/OL.43.003846DOI Listing
August 2018

Noninvasive Optical Assessment of Implanted Engineered Tissues Correlates with Cytokine Secretion.

Tissue Eng Part C Methods 2018 04;24(4):214-221

1 Department of Biomedical Engineering, College of Engineering & Medical School, University of Michigan , Ann Arbor, Michigan.

Fluorescence lifetime sensing has been shown to noninvasively characterize the preimplantation health and viability of engineered tissue constructs. However, current practices to monitor postimplantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). We employed label-free fluorescence lifetime spectroscopy for quantitative, noninvasive optical assessment of engineered tissue constructs that were implanted into a murine model. The portable system was designed to be suitable for intravital measurements and included a handheld probe to precisely and rapidly acquire data at multiple sites per construct. Our model tissue constructs were manufactured from primary human cells to simulate patient variability based on a standard protocol, and half of the manufactured constructs were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess preimplantation construct health: interleukin-8 (IL-8), human β-defensin 1 (hBD-1), and vascular endothelial growth factor (VEGF). Preimplantation cytokine secretion ranged from 1.5 to 33.5 pg/mL for IL-8, from 3.4 to 195.0 pg/mL for hBD-1, and from 0.1 to 154.3 pg/mL for VEGF. In vivo optical sensing assessed constructs at 1 and 3 weeks postimplantation. We found that at 1 week postimplantation, in vivo optical parameters correlated with in vitro preimplantation secretion levels of all three cytokines (p < 0.05). This correlation was not observed in optical measurements at 3 weeks postimplantation when histology showed that the constructs had re-epithelialized, independent of preimplantation health state, supporting the lack of a correlation. These results suggest that clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor postimplantation integration of engineered tissues.
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http://dx.doi.org/10.1089/ten.TEC.2017.0516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905861PMC
April 2018

Compact dual-mode diffuse optical system for blood perfusion monitoring in a porcine model of microvascular tissue flaps.

J Biomed Opt 2017 Dec;22(12):1-14

University of Michigan, Department of Biomedical Engineering, Ann Arbor, Michigan, United States.

In reconstructive surgery, the ability to detect blood flow interruptions to grafted tissue represents a critical step in preventing postsurgical complications. We have developed and pilot tested a compact, fiber-based device that combines two complimentary modalities-diffuse correlation spectroscopy (DCS) and diffuse reflectance spectroscopy-to quantitatively monitor blood perfusion. We present a proof-of-concept study on an in vivo porcine model (n=8). With a controllable arterial blood flow supply, occlusion studies (n=4) were performed on surgically isolated free flaps while the device simultaneously monitored blood flow through the supplying artery as well as flap perfusion from three orientations: the distal side of the flap and two transdermal channels. Further studies featuring long-term monitoring, arterial failure simulations, and venous failure simulations were performed on flaps that had undergone an anastomosis procedure (n=4). Additionally, benchtop verification of the DCS system was performed on liquid flow phantoms. Data revealed relationships between diffuse optical measures and state of occlusion as well as the ability to detect arterial and venous compromise. The compact construction of the device, along with its noninvasive and quantitative nature, would make this technology suitable for clinical translation.
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http://dx.doi.org/10.1117/1.JBO.22.12.121609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729962PMC
December 2017

Quantitative, Label-Free Evaluation of Tissue-Engineered Skeletal Muscle Through Multiphoton Microscopy.

Tissue Eng Part C Methods 2017 10 20;23(10):616-626. Epub 2017 Sep 20.

1 Department of Biomedical Engineering, University of Michigan , Ann Arbor, Michigan.

The lack of tools for assessing engineered tissue viability and function in a noninvasive manner is a major regulatory and translational challenge facing tissue engineers. Label-free, nonlinear optical molecular imaging (OMI) has utilized endogenous nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence to indicate metabolic activity. Similarly, second harmonic generation (SHG) signals from myosin and collagen can measure overall muscle structural integrity and function. The purpose of this study was to demonstrate these OMI techniques for the first time in engineered skeletal muscle and to develop a novel method for evaluating our engineered skeletal muscle units (SMUs) before implantation. Three experimental groups were studied: Control, Steroid Supplemented, and Metabolically Stressed SMUs. After imaging and analysis in ImageJ, a redox ratio (RR) metric was calculated to indicate metabolic activity, and a structure ratio metric was calculated to reflect structural composition. In addition, function was evaluated as tetanic force production in response to electrical stimulation. In living tissues, the RRs successfully distinguished control and metabolically stressed SMUs in both monolayer and 3D form. OMI of myosin and collagen SHG similarly differentiated control SMUs from the steroid supplemented group. With respect to function, steroid supplementation significantly increased active force generation. When comparing functional and OMI measures, a significant correlation was present between overall myosin density and active force generation. This work demonstrates the potential for using label-free OMI to evaluate tissue-engineered skeletal muscle constructs. The positive correlation between structural OMI measures and force production suggests that OMI could potentially serve as an accurate predictor of functional behaviors, such as integration and tissue regeneration, after implantation. This noninvasive OMI methodology, demonstrated for the first time in engineered skeletal muscle, could prove invaluable for assessing our tissue engineering technology and confirming release criteria for validation.
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http://dx.doi.org/10.1089/ten.TEC.2017.0284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653135PMC
October 2017

Design verification of a compact system for detecting tissue perfusion using bimodal diffuse optical technologies.

Proc SPIE Int Soc Opt Eng 2017 Jan-Feb;10072. Epub 2017 Feb 17.

University of Michigan (United States).

It is essential to monitor tissue perfusion during and after reconstructive surgery, as restricted blood flow can result in graft failures. Current clinical procedures are insufficient to monitor tissue perfusion, as they are intermittent and often subjective. To address this unmet clinical need, a compact, low-cost, multimodal diffuse correlation spectroscopy and diffuse reflectance spectroscopy system was developed. We verified system performance via tissue phantoms and experimental protocols for rigorous bench testing. Quantitative data analysis methods were employed and tested to enable the extraction of tissue perfusion parameters. This design verification study assures data integrity in future studies.
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http://dx.doi.org/10.1117/12.2252811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946689PMC
February 2017

preclinical verification of a multimodal diffuse reflectance and correlation spectroscopy system for sensing tissue perfusion.

Proc SPIE Int Soc Opt Eng 2017 Jan-Feb;10072. Epub 2017 Feb 17.

Applied Physics Program, University of Michigan, Ann Arbor, MI 48109.

In reconstructive surgery, impeded blood flow in microvascular free flaps due to a compromise in arterial or venous patency secondary to blood clots or vessel spasms can rapidly result in flap failures. Thus, the ability to detect changes in microvascular free flaps is critical. In this paper, we report progress on pre-clinical testing of a compact, multimodal, fiber-based diffuse correlation and reflectance spectroscopy system designed to quantitatively monitor tissue perfusion in a porcine model's surgically-grafted free flap. We also describe the device's sensitivity to incremental blood flow changes and discuss the prospects for continuous perfusion monitoring in future clinical translational studies.
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http://dx.doi.org/10.1117/12.2252620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916836PMC
February 2017

Novel diffuse optics system for continuous tissue viability monitoring - extended recovery testing in a porcine flap model.

Proc SPIE Int Soc Opt Eng 2017 Jan-Feb;10054. Epub 2017 Feb 14.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109.

In reconstructive surgery, tissue perfusion/vessel patency is critical to the success of microvascular free tissue flaps. Early detection of flap failure secondary to compromise of vascular perfusion would significantly increase the chances of flap salvage. We have developed a compact, clinically-compatible monitoring system to enable automated, minimally-invasive, continuous, and quantitative assessment of flap viability/perfusion. We tested the system's continuous monitoring capability during extended non-recovery surgery using an porcine free flap model. Initial results indicated that the system could assess flap viability/perfusion in a quantitative and continuous manner. With proven performance, the compact form constructed with cost-effective components would make this system suitable for clinical translation.
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http://dx.doi.org/10.1117/12.2252295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916821PMC
February 2017

Tissue Classification Using Optical Spectroscopy Accurately Differentiates Cancer and Chronic Pancreatitis.

Pancreas 2017 02;46(2):244-251

From the *Department of Biomedical Engineering, †Department of Internal Medicine, ‡Comprehensive Cancer Center §Department of Biostatistics, ∥Department of Pathology, and ¶Department of Surgery, University of Michigan, Ann Arbor, MI.

Objectives: Current pancreatic cancer diagnostics cannot reliably detect early disease or distinguish it from chronic pancreatitis. We test the hypothesis that optical spectroscopy can accurately differentiate cancer from chronic pancreatitis and normal pancreas. We developed and tested clinically compatible multimodal optical spectroscopy technology to measure reflectance and endogenous fluorescence from human pancreatic tissues.

Methods: Freshly excised pancreatic tissue specimens (39 normal, 34 chronic pancreatitis, 32 adenocarcinoma) from 18 patients were optically interrogated, with site-specific histopathology representing the criterion standard. A multinomial logistic model using principal component analysis and generalized estimating equations provided statistically rigorous tissue classification.

Results: Optical spectroscopy distinguished pancreatic cancer from normal pancreas and chronic pancreatitis (sensitivity, 91%; specificity, 82%; positive predictive value, 69%; negative predictive value, 95%; area under receiver operating characteristic curve, 0.89). Reflectance alone provided essentially the same classification accuracy as reflectance and fluorescence combined, suggesting that a rapid, low-cost, reduced-footprint, reflectance-based device could be deployed without notable loss of diagnostic power.

Conclusions: Our novel, clinically compatible, label-free optical diagnostic technology accurately characterizes pancreatic tissues. These data provide the scientific foundation demonstrating that optical spectroscopy can potentially improve diagnosis of pancreatic cancer and chronic pancreatitis.
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http://dx.doi.org/10.1097/MPA.0000000000000732DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5235923PMC
February 2017

A compact instrument to measure perfusion of vasculature in transplanted maxillofacial free flaps.

Proc SPIE Int Soc Opt Eng 2016 Feb 4;9715. Epub 2016 Mar 4.

Radiation Monitoring Devices, Inc., 44 Hunt St., Watertown, MA, 02472 USA.

The vascularization and resulting perfusion of transferred tissues are critical to the success of grafts in buried free flap transplantations. To enable long-term clinical monitoring of grafted tissue perfusion during neovascularization and endothelialization, we are developing an implantable instrument for the continuous monitoring of perfusion using diffuse correlation spectroscopy (DCS), and augmented with diffuse reflectance spectroscopy (DRS). This work discusses instrument construction, integration, and preliminary results using a porcine graft model.
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http://dx.doi.org/10.1117/12.2212872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916819PMC
February 2016

A disposable, flexible skin patch for clinical optical perfusion monitoring at multiple depths.

Proc SPIE Int Soc Opt Eng 2016 Feb 4;9715. Epub 2016 Mar 4.

Department of Emergency Medicine, Dartmouth-Hitchcock Medical, Lebanon, NH 03766.

Stable, relative localization of source and detection fibers is necessary for clinical implementation of quantitative optical perfusion monitoring methods such as diffuse correlation spectroscopy (DCS) and diffuse reflectance spectroscopy (DRS). A flexible and compact device design is presented as a platform for simultaneous monitoring of perfusion at a range of depths, enabled by precise location of optical fibers in a robust and secure adhesive patch. We will discuss preliminary data collected on human subjects in a lower body negative pressure model for hypovolemic shock. These data indicate that this method facilitates simple and stable simultaneous monitoring of perfusion at multiple depths and within multiple physiological compartments.
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http://dx.doi.org/10.1117/12.2230988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647776PMC
February 2016

Optical methods for quantitative and label-free sensing in living human tissues: principles, techniques, and applications.

Adv Phys 2016 1;1(4):523-543. Epub 2016 Sep 1.

Department of Biomedical Engineering, Applied Physics Program, University of Michigan, Ann Arbor, MI, USA.

We present an overview of quantitative and label-free optical methods used to characterize living biological tissues, with an emphasis on emerging applications in clinical tissue diagnostics. Specifically, this review focuses on diffuse optical spectroscopy, imaging, and tomography, optical coherence-based techniques, and non-linear optical methods for molecular imaging. The potential for non- or minimally-invasive assessment, quantitative diagnostics, and continuous monitoring enabled by these tissue-optics technologies provides significant promise for continued clinical translation.
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http://dx.doi.org/10.1080/23746149.2016.1221739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560608PMC
September 2016

FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy.

Mol Reprod Dev 2015 Jul-Aug;82(7-8):587-604. Epub 2015 May 25.

Department of Bioengineering, University of California, Riverside, California.

The combination of fluorescent-probe technology plus modern optical microscopes allows investigators to monitor dynamic events in living cells with exquisite temporal and spatial resolution. Fluorescence recovery after photobleaching (FRAP), for example, has long been used to monitor molecular dynamics both within cells and on cellular surfaces. Although bound by the diffraction limit imposed on all optical microscopes, the combination of digital cameras and the application of fluorescence intensity information on large-pixel arrays have allowed such dynamic information to be monitored and quantified. Fluorescence lifetime imaging microscopy (FLIM), on the other hand, utilizes the information from an ensemble of fluorophores to probe changes in the local environment. Using either fluorescence-intensity or lifetime approaches, fluorescence resonance energy transfer (FRET) microscopy provides information about molecular interactions, with Ångstrom resolution. In this review, we summarize the theoretical framework underlying these methods and illustrate their utility in addressing important problems in reproductive and developmental systems.
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http://dx.doi.org/10.1002/mrd.22501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515154PMC
April 2016

The potential of label-free nonlinear optical molecular microscopy to non-invasively characterize the viability of engineered human tissue constructs.

Biomaterials 2014 Aug 20;35(25):6667-76. Epub 2014 May 20.

Department of Biomedical Engineering, University of Michigan College of Engineering & Medical School, 1101 Beal Avenue, Ann Arbor, MI 48109-2110, USA. Electronic address:

Nonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine.
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http://dx.doi.org/10.1016/j.biomaterials.2014.04.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106121PMC
August 2014

Introduction: feature issue on optical molecular probes, imaging, and drug delivery.

Biomed Opt Express 2014 Feb 29;5(2):643-4. Epub 2014 Jan 29.

Department of Biomedical Engineering, Applied Physics Program, Univ. of Michigan, Ann Arbor MI 48109, USA.

The editors introduce the Biomedical Optics Express feature issue "Optical Molecular Probes, Imaging, and Drug Delivery," which is associated with a Topical Meeting of the same name held at the 2013 Optical Society of America (OSA) Optics in the Life Sciences Congress in Waikoloa Beach, Hawaii, April 14-18, 2013. The international meeting focused on the convergence of optical physics, photonics technology, nanoscience, and photochemistry with drug discovery and clinical medicine. Papers in this feature issue are representative of meeting topics, including advances in microscopy, nanotechnology, and optics in cancer research.
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http://dx.doi.org/10.1364/BOE.5.000643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920892PMC
February 2014

In vivo optical spectroscopy for improved detection of pancreatic adenocarcinoma: a feasibility study.

Biomed Opt Express 2013 Dec 2;5(1):9-15. Epub 2013 Dec 2.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109-2099, USA ; Applied Physics Program, University of Michigan, Ann Arbor, MI 48109-1040, USA ; Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109-0944, USA.

Pancreatic adenocarcinoma has a five-year survival rate of less than 6%. This low survival rate is attributed to the lack of accurate detection methods, which limits diagnosis to late-stage disease. Here, an in vivo pilot study assesses the feasibility of optical spectroscopy to improve clinical detection of pancreatic adenocarcinoma. During surgery on 6 patients, we collected spectrally-resolved reflectance and fluorescence in vivo. Site-matched in vivo and ex vivo data agreed qualitatively and quantitatively. Quantified differences between adenocarcinoma and normal tissues in vivo were consistent with previous results from a large ex vivo data set. Thus, optical spectroscopy is a promising method for the improved diagnosis of pancreatic cancer in vivo.
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http://dx.doi.org/10.1364/BOE.5.000009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891348PMC
December 2013

Characterizing human pancreatic cancer precursor using quantitative tissue optical spectroscopy.

Biomed Opt Express 2013 14;4(12):2828-34. Epub 2013 Nov 14.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109-2099, USA ; Applied Physics Program, University of Michigan, Ann Arbor, MI 48109-1040, USA ; Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109-0944, USA.

In a pilot study, multimodal optical spectroscopy coupled with quantitative tissue-optics models distinguished intraductal papillary mucinous neoplasm (IPMN), a common precursor to pancreatic cancer, from normal tissues in freshly excised human pancreas. A photon-tissue interaction (PTI) model extracted parameters associated with cellular nuclear size and refractive index (from reflectance spectra) and extracellular collagen content (from fluorescence spectra). The results suggest that tissue optical spectroscopy has the potential to characterize pre-cancerous neoplasms in human pancreatic tissues.
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http://dx.doi.org/10.1364/BOE.4.002828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3862164PMC
January 2014

Characterization of squamous cell carcinoma in an organotypic culture via subsurface non-linear optical molecular imaging.

Exp Biol Med (Maywood) 2013 Nov 1;238(11):1233-41. Epub 2013 Oct 1.

Department of Periodontics and Oral Medicine, School of Dentistry, University of Michigan, MI 48109-1078, USA.

Tristetraprolin (TTP) is an RNA-binding protein which downregulates multiple cytokines that mediate progression of head and neck squamous cell carcinoma (HNSCC). We previously showed that HNSCC cells with shRNA-mediated knockdown of TTP are more invasive than controls. In this study, we use control and TTP-deficient cells to present a novel subsurface non-linear optical molecular imaging method using a three-dimensional (3D) organotypic construct, and compare the live cell imaging data to histology of fixed tissue specimens. This manuscript describes how to prepare and image the novel organotypic system that closely mimics HNSCC in a clinical setting. The oral cancer equivalent (OCE) system allows HNSCC cells to stratify and invade beyond the basement membrane into underlying connective tissue prepared from decellularized human dermal tissue. The OCE model was inspired by tissue engineering strategies to prepare autologous transplants from human keratinocytes. Advantages of this method over previously used in vitro cancer models include the simulation of the basement membrane and complex connective tissue in the construct, in addition to the ability to track the 3D movement of live invading cells and quantify matrix destruction over time. The OCE model and novel live cell imaging strategy may be applied to study other types of 3D tissue constructs.
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http://dx.doi.org/10.1177/1535370213502628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216589PMC
November 2013

Fluorescence lifetime imaging microscopy for quantitative biological imaging.

Methods Cell Biol 2013 ;114:457-88

Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA.

Fluorescence lifetime imaging microscopy (FLIM) is a method for measuring fluorophore lifetimes with microscopic spatial resolution, providing a useful tool for cell biologists to detect, visualize, and investigate structure and function of biological systems. In this chapter, we begin by introducing the basic theory of fluorescence lifetime, including the characteristics of fluorophore decay, followed by a discussion of factors affecting fluorescence lifetimes and the potential advantages of fluorescence lifetime as a source of image contrast. Experimental methods for creating lifetime maps, including both time- and frequency-domain experimental approaches, are then introduced. Then, FLIM data analysis methods are discussed, including rapid lifetime determination, multiexponential fitting, Laguerre polynomial fitting, and phasor plot analysis. After, data analysis methods are introduced that improve lifetime precision of FLIM maps based upon optimal virtual gating and total variation denoising. The chapter concludes by highlighting several recent FLIM applications for quantitative biological imaging, including Förster resonance energy transfer-FLIM, fluorescence correlation spectroscopy-FLIM, multispectral-FLIM, and multiphoton-FLIM.
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http://dx.doi.org/10.1016/B978-0-12-407761-4.00020-8DOI Listing
February 2014

Total variation versus wavelet-based methods for image denoising in fluorescence lifetime imaging microscopy.

J Biophotonics 2012 May 13;5(5-6):449-57. Epub 2012 Mar 13.

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109-2099, USA.

We report the first application of wavelet-based denoising (noise removal) methods to time-domain box-car fluorescence lifetime imaging microscopy (FLIM) images and compare the results to novel total variation (TV) denoising methods. Methods were tested first on artificial images and then applied to low-light live-cell images. Relative to undenoised images, TV methods could improve lifetime precision up to 10-fold in artificial images, while preserving the overall accuracy of lifetime and amplitude values of a single-exponential decay model and improving local lifetime fitting in live-cell images. Wavelet-based methods were at least 4-fold faster than TV methods, but could introduce significant inaccuracies in recovered lifetime values. The denoising methods discussed can potentially enhance a variety of FLIM applications, including live-cell, in vivo animal, or endoscopic imaging studies, especially under challenging imaging conditions such as low-light or fast video-rate imaging.
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http://dx.doi.org/10.1002/jbio.201100137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106132PMC
May 2012

Development of a spatially offset Raman spectroscopy probe for breast tumor surgical margin evaluation.

J Biomed Opt 2011 Jul;16(7):077006

Vanderbilt University, Department of Biomedical Engineering, Nashville, Tennessee 37235, USA.

The risk of local recurrence for breast cancers is strongly correlated with the presence of a tumor within 1 to 2 mm of the surgical margin on the excised specimen. Previous experimental and theoretical results suggest that spatially offset Raman spectroscopy (SORS) holds much promise for intraoperative margin analysis. Based on simulation predictions for signal-to-noise ratio differences among varying spatial offsets, a SORS probe with multiple source-detector offsets was designed and tested. It was then employed to acquire spectra from 35 frozen-thawed breast tissue samples in vitro. Spectra from each detector ring were averaged to create a composite spectrum with biochemical information covering the entire range from the tissue surface to ∼2 mm below the surface, and a probabilistic classification scheme was used to classify these composite spectra as "negative" or "positive" margins. This discrimination was performed with 95% sensitivity and 100% specificity, or with 100% positive predictive value and 94% negative predictive value.
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http://dx.doi.org/10.1117/1.3600708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144975PMC
July 2011

Instrumentation to rapidly acquire fluorescence wavelength-time matrices of biological tissues.

Biomed Opt Express 2010 Aug 10;1(2):574-586. Epub 2010 Aug 10.

A fiber-optic system was developed to rapidly acquire tissue fluorescence wavelength-time matrices (WTMs) with high signal-to-noise ratio (SNR). The essential system components (473 nm microchip laser operating at 3 kHz repetition frequency, fiber-probe assemblies, emission monochromator, photomultiplier tube, and digitizer) were assembled into a compact and clinically-compatible unit. Data were acquired from fluorescence standards and tissue-simulating phantoms to test system performance. Fluorescence decay waveforms with SNR > 100 at the decay curve peak were obtained in less than 30 ms. With optimized data transfer and monochromator stepping functions, it should be feasible to acquire a full WTM at 5 nm emission wavelength intervals over a 200 nm range in under 2 seconds.
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http://dx.doi.org/10.1364/BOE.1.000574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018017PMC
August 2010

Enhancing precision in time-domain fluorescence lifetime imaging.

J Biomed Opt 2010 Sep-Oct;15(5):056013

University of Michigan, Department of Biomedical Engineering, Ann Arbor, Michigan 48109-2099, USA.

In biological applications of fluorescence lifetime imaging, low signals from samples can be a challenge, causing poor lifetime precision. We demonstrate how optimal signal gating (a method applied to the temporal dimension of a lifetime image) and novel total variation denoising models (a method applied to the spatial dimension of a lifetime image) can be used in time-domain fluorescence lifetime imaging microscopy (FLIM) to improve lifetime precision. In time-gated FLIM, notable fourfold precision improvements were observed in a low-light example. This approach can be employed to improve FLIM data while minimizing sample light exposure and increasing imaging speed.
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http://dx.doi.org/10.1117/1.3494566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966491PMC
March 2011

Photon-tissue interaction model enables quantitative optical analysis of human pancreatic tissues.

Opt Express 2010 Oct;18(21):21612-21

Applied Physics Program, University of Michigan, Ann Arbor, MI 48109-1040, USA.

A photon-tissue interaction (PTI) model was developed and employed to analyze 96 pairs of reflectance and fluorescence spectra from freshly excised human pancreatic tissues. For each pair of spectra, the PTI model extracted a cellular nuclear size parameter from the measured reflectance, and the relative contributions of extracellular and intracellular fluorophores to the intrinsic fluorescence. The results suggest that reflectance and fluorescence spectroscopies have the potential to quantitatively distinguish among pancreatic tissue types, including normal pancreatic tissue, pancreatitis, and pancreatic adenocarcinoma.
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http://dx.doi.org/10.1364/OE.18.021612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408914PMC
October 2010

Quantitative polarized Raman spectroscopy in highly turbid bone tissue.

J Biomed Opt 2010 May-Jun;15(3):037001

University of Michigan, Department of Biomedical Engineering, 930 North University Avenue, Room 4638, Ann Arbor, Michigan 48109, USA.

Polarized Raman spectroscopy allows measurement of molecular orientation and composition and is widely used in the study of polymer systems. Here, we extend the technique to the extraction of quantitative orientation information from bone tissue, which is optically thick and highly turbid. We discuss multiple scattering effects in tissue and show that repeated measurements using a series of objectives of differing numerical apertures can be employed to assess the contributions of sample turbidity and depth of field on polarized Raman measurements. A high numerical aperture objective minimizes the systematic errors introduced by multiple scattering. We test and validate the use of polarized Raman spectroscopy using wild-type and genetically modified (oim/oim model of osteogenesis imperfecta) murine bones. Mineral orientation distribution functions show that mineral crystallites are not as well aligned (p<0.05) in oim/oim bones (28+/-3 deg) compared to wild-type bones (22+/-3 deg), in agreement with small-angle X-ray scattering results. In wild-type mice, backbone carbonyl orientation is 76+/-2 deg and in oim/oim mice, it is 72+/-4 deg (p>0.05). We provide evidence that simultaneous quantitative measurements of mineral and collagen orientations on intact bone specimens are possible using polarized Raman spectroscopy.
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http://dx.doi.org/10.1117/1.3426310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881928PMC
October 2010

Precise fluorophore lifetime mapping in live-cell, multi-photon excitation microscopy.

Opt Express 2010 Apr;18(8):8688-96

Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan 48109-2099, USA.

Fluorophore excited state lifetime is a useful indicator of micro-environment in cellular optical molecular imaging. For quantitative sensing, precise lifetime determination is important, yet is often difficult to accomplish when using the experimental conditions favored by live cells. Here we report the first application of temporal optimization and spatial denoising methods to two-photon time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to improve lifetime precision in live-cell images. The results demonstrated a greater than five-fold improvement in lifetime precision. This approach minimizes the adverse effects of excitation light on live cells and should benefit FLIM applications to high content analysis and bioimage informatics.
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http://dx.doi.org/10.1364/OE.18.008688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410727PMC
April 2010
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