This PDF file contains the front matter associated with SPIE Proceedings Volume 8719, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

Bacteria use an intricate set of communication systems for sensing and interpreting environmental cues that coordinate population-based behavior. Quorum sensing is one of these systems, and it involves the production, release, and detection of small chemical signaling molecules. Recent research has revealed the role of quorum sensing molecules in the control of microbial activities such as biofilm formation. In this presentation we outline the development of a recombinant E. coli cell-based sensor for detection of the quorum sensing molecule Autoinducer-2 (AI-2), as well as engineering strategies to remove sugar and anoxic inhibition of the strain.

Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

As software and methodology develop, key aspects of molecular interactions such as detailed energetics and flexibility are continuously better represented in docking simulations. In the latest iteration of the XPairIt API and Docking Protocol, we perform a blind dock of a peptide into the cleavage site of the Anthrax lethal factor (LF) metalloprotein. Molecular structures are prepared from RCSB:1JKY and we demonstrate a reasonably accurate docked peptide through analysis of protein motion and, using NCI Plot, visualize and characterize the forces leading to binding. We compare our docked structure to the 1JKY crystal structure and the more recent 1PWV structure, and discuss both captured and overlooked interactions. Our results offer a more detailed look at secondary contact and show that both van der Waals and electrostatic interactions from peptide residues further from the enzyme's catalytic site are significant.

Lab-on-a-chip (LoC) systems translating the whole process of pathogen analysis to an integrated, miniaturized, and automatically functioning microfluidic platform are generally expected to be very promising future diagnostic approaches. The development of such a LoC system for the detection of bacterial pathogens applied to the example pathogen Francisella tularensis is described in this report. To allow functional testing of the whole process cascade before final device integration, various bio-analytical steps such as cell lysis, DNA extraction and purification, continuous-flow PCR and analyte detection have been adapted to unique functional microfluidic modules. As a successive step, positively tested modules for pathogen detection have been successfully assembled to an integrated chip. Moreover, technical solutions for a smooth interaction between sample input from the outer world as well as microfluidic chip and chip driving instrument have been developed. In conclusion, a full repertoire of analytical tools have been developed and successfully tested in the concerted manner of a functionally integrated microfluidic device representing a tool for future diagnostic approaches.

Optical detection methods have been implemented on micro-fluidic chips containing channels or cavities of different geometries e.g. for colorimetry or fluorescence measurements with excitation within the chip plane [1-2]. The most prominent problem of the read-out from a micro-fluidic chip is the limitation of the optical yield. Without e.g. an immersion liquid for compensation of the total reflection on the boundary, only about 11-13% of rays cross over the boundary from a polymer chip to air. One efficient method to increase the optical yield from a chip is a ray reorientation inside of the chip using an additional surface structure creating new incident refraction conditions on the boundary before rays are leaving the chip. The use of 45°-tilted mirror arrangements for in- and out-coupling of the fluorescence signal from a micro-fluidic chip and the realization of this principle for low-cost fluorescence detection systems have been published [3].

This paper includes the investigation of the effect of different tilt angles of total reflection and metallized-surface mirrors for an analyte volume emitter, using the ray-tracing simulation tool OptiCAD10. Furthermore, an estimation of the influence of a surface-emitted signal for different geometries of metallized detection cells with or without a combination with external lenses on the out-coupling efficiency will be presented. The best result of an out-coupling efficiency increase of 10 times was achieved for a combination of a structured and metallized detection cell with an external cylindrical lens.

Aim of the study was to establish and evaluate a “lab-on-a-chip” system for the detection of bacterial B-agents using Bacillus (B.) thuringiensis as simulant for B. anthracis.

To enable reliable detection of target DNA using PCR assays it is crucial that purified DNA is extracted from the sample matrix. We established chip-based assays for cell lysis, sample concentration, and DNA purification using magnetic particles with special surface modifications and compared these assays with a commercial routine method. DNA yield was determined using quantitative real-time PCR assays with TaqMan probes targeting the cry1Ac gene.

Lab-on-a-chip systems are applicable for point-of-care analysis and provide several advantages in comparison to conventional diagnostic techniques. Purification of DNA and subsequent PCR analysis can be integrated and the instrumentation can be miniaturized. Therefore, such tests can also be useful in medical and veterinary diagnostics.

Using the Navier-Stokes equation and assuming a viscosity radially modulated for a quasi-Newtonian fluid, we obtain the impedance of a fluid through microchannels and their corresponding electrical analogs. To solve the Navier-Stokes equation will use the Laplace transform, the Bromwich integral, the residue theorem and Bessel functions. This will give a formula for the impedance in terms of Bessel functions and from these equations to be constructed equivalent electrical circuits. These solutions correspond to the case of quasi-Newtonian fluid it is to say a fluid that does not stagnate in the channel wall as is the case if the fluid is Newtonian. The formulas obtained may have applications in the general theory of microfluidics and microscopic systems design for drug delivery.

A cylindrical matrix device with a circular release area with inhomogeneous diffusivity was analyzed using a Laplace transform–based method, using Bromwich integral and residue theorem. The two-dimensional model represented a pharmaceutical agent uniformly distributed in a polymeric matrix with a diffusivity spatially modulated, surrounded by an impermeable layer. The pharmaceutical agent could be transferred only through a small hole centered at the top surface of the cylinder. A closed-form solution was obtained in terms of Bessel functions with the aim to help study the effects of design parameters and geometries on the cumulative amount of pharmaceutical agent released. The cumulative flux of pharmaceutical agent increased with the mass transfer and diffusion coefficients and decreased with any increment in the device’s length and variations of the diffusivity coefficients. The delivery rate was described by an effective time constant calculated from Laplace transforms and using Bessel functions and their zeros. Reducing the orifice diameter or fabricating a longer system would delay transport of the medication. Simplified expressions for the release profile and the time constant were derived for special design cases.

Advances in biocompatible materials and electrocatalytic nanomaterials have extended and enhanced the field of biosensors. Immobilization of biorecognition elements on nanomaterial platforms is an efficient technique for developing high fidelity biosensors. Single layer (i.e., Langmuir–Blodgett) protein films are efficient, but disadvantages of this approach include high cost, mass transfer limitations, and Vromer competition for surface binding sites. There is a need for simple, user friendly protein-nanomaterial sensing membranes that can be developed in laboratories or classrooms (i.e., outside of the clean room). In this research, we develop high fidelity nanomaterial platforms for developing electrochemical biosensors using sustainable biomaterials and user-friendly deposition techniques. Catalytic nanomaterial platforms are developed using a combination of self assembled monolayer chemistry and electrodeposition. High performance biomaterials (e.g., nanolignin) are recovered from paper pulp waste and combined with proteins and nanomaterials to form active sensor membranes. These methods are being used to develop electrochemical biosensors for studying physiological transport in biomedical, agricultural, and environmental applications.

Electrochemical-aptamer based (E-AB) sensors represent a universal specific, selective, and sensitive sensing platform for the detection of small molecule targets. Their specific detection abilities are afforded by oligonucleotide (RNA or DNA) aptamers employed as electrode-bound biorecognition elements. Sensor signaling is predicated on bindinginduced changes in conformation and/or flexibility of the aptamer that is readily measurable electrochemically. While sensors fabricated using DNA aptamers can achieve specific and selective detection even in unadulterated sample matrices, such as blood serum, RNA-based sensors fail when challenged in the same sample matrix without significant sample pretreatment. This failure is at least partially a result of enzymatic degradation of the RNA sensing element. This degradation destroys the sensing aptamer inhibiting the quantitative measurement of the target analyte and thus limits the application of E-AB sensors constructed with RNA aptamer. To circumvent this, we demonstrate that a biocompatible hydrogel membrane protects the RNA aptamer sensor surface from enzymatic degradation for at least 3 hours - a remarkable improvement over the rapid (~minutes) degradation of unprotected sensors. To demonstrate this, we characterize the response of sensors fabricated with representative DNA and RNA aptamers directed against the aminoglycoside antibiotic, tobramycin in blood serum both protected and unprotected by a polyacrylamide membrane. Furthermore, we find encapsulation of the sensor surface with the hydrogel does not significantly impede the detection ability of aptamer-based sensors. This hydrogel-aptamer interface will thus likely prove useful for the long-term monitoring of therapeutics in complex biological media.

This paper proposes a novel technique for utilizing electrically conductive textiles as a distributed sensor for detecting and localizing liquids (e.g., blood), damage (e.g., rips, cuts, bullet holes) and, potentially, biosignals. The proposed technique is verified through both simulation and experimental measurements. Circuit theory is employed to depict conductive fabric as a bounded, near-infinite grid of resistors. Solutions to the well-known infinite resistance grid problem are used to confirm the accuracy and validity of this modeling approach. Simulations allow for discontinuities to be placed within the resistor matrix to illustrate the effects of bullet holes within the fabric. A real-time experimental system was developed that uses a multiplexed, Wheatstone bridge measurement approach to determine the resistances of a coarse electrode grid across the conductive fabric. Non-uniform resistance values of the grid infer the presence of liquids and rips in the fabric. The resistor-grid model is validated through a comparison of simulated and experimental results. Results suggest accuracy proportional to the electrode spacing in determining the presence and location of disturbances in conductive fabric samples. Future work is focused on refining the experimental system to provide more accuracy in detecting and localizing events (although just the knowledge of a penetration may be adequate for some intended applications) as well as developing a complete prototype that can be deployed for field testing. Potential applications include intelligent clothing, flexible, lightweight sensing systems, and combat wound detection.

Non-invasive mechanical property estimation of an embedded object (tumor) can be used in medicine for characterization between malignant and benign lesions. We developed a tactile imaging sensor which is capable of detecting mechanical properties of inclusions. Studies show that stiffness of tumor is a key physiological discerning parameter for malignancy. As our sensor compresses the tumor from the surface, the sensing probe deforms, and the light scatters. This forms the tactile image. Using the features of the image, we can estimate the mechanical properties such as size, depth, and elasticity of the embedded object. To test the performance of the method, a phantom study was performed. Silicone rubber balls were used as embedded objects inside the tissue mimicking substrate made of Polydimethylsiloxane. The average relative errors for size, depth, and elasticity were found to be 67.5%, 48.2%, and 69.1%, respectively. To test the feasibility of the sensor in estimating the elasticity of tumor, a pilot clinical study was performed on twenty breast cancer patients. The estimated elasticity was correlated with the biopsy results. Preliminary results show that the sensitivity of 67% and the specificity of 91.7% for elasticity. Results from the clinical study suggest that the tactile imaging sensor may be used as a tumor malignancy characterization tool.

Engagement monitoring is crucial in many clinical and therapy applications such as early learning preschool classes for children with developmental delays including autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD), or cerebral palsy; as it is challenging for the instructors to evaluate the individual responses of these children to determine the effectiveness of the teaching strategies due to the diverse and unique need of each child who might have difficulty in verbal or behavioral communication. This paper presents an ambulatory scalp electroencephalogram (EEG) NeuroMonitor platform to study brain engagement activities in natural settings. The developed platform is miniature (size: 2.2” x 0.8” x 0.36”, weight: 41.8 gm with 800 mAh Li-ion battery and 3 snap leads) and low-power (active mode: 32 mA low power mode: under 5mA) with 2 channels (Fp1, Fp2) to record prefrontal cortex activities of the subject in natural settings while concealed within a headband. The signals from the electrodes are amplified with a low-power instrumentation amplifier; notch filtered (f_c = 60Hz), then band-passed by a 2nd-order Chebyshev-I low-pass filter cascaded with a 2nd-order low-pass (f_c = 125Hz). A PSoC ADC (16-bit, 256 sps) samples this filtered signal, and can either transmit it through a Class-2 Bluetooth transceiver to a remote station for real-time analysis or store it in a microSD card for offline processing. This platform is currently being evaluated to capture data in the classroom settings for engagement monitoring of children, aimed to study the effectiveness of various teaching strategies that will allow the development of personalized classroom curriculum for children with developmental delays.

Using a 94-GHz homodyne interferometer employing a highly-directional quasi-optical lens antenna aimed at a human subject's chest, we can measure chest wall displacement from up to 10m away and through common clothing. Within the chest displacement signal are motions due to cardiac activity, respiration, and gross body movement. Our goal is to find the heart rate of the subject being monitored, which implies isolation of the minute movements due to cardiac activity from the much larger movements due to respiration and body movement. To accomplish this, we first find a subset of the true heartbeat temporal locations (called confident" heartbeats) in the displacement signal using a multi-resolution wavelet approach, utilizing Symlet wavelets. Although the chest displacement due to cardiac activity is orders of magnitude smaller than that due to respiration and body movement, wavelets find those heartbeat locations due to several useful properties, such as shape matching, high-pass filtering, and vanishing moments. Using the assumption that the confident" heartbeats are randomly selected from the set of all heartbeats, we are able to find the maximum a posteriori statistics of an inverse Gaussian probability distribution modeling the inter-heartbeat times. We then analyze the confident" heartbeats and decide which heartbeats are probabilistically correct and which are not, based on the inverse Gaussian distribution we calculated earlier. The union of the confident" set, after pruning, and the interpolated set forms a very close approximation to the true heartbeat temporal location set, and thus allows us to accurately calculate a heart rate.

Non-invasive tools that allow real-time quantification of molecules relevant to metabolism, homeostasis, and cell signaling in cells and tissue are of great importance for studying physiology. Several microsensor technologies have been developed to monitor concentration of molecules such as ions, oxygen, electroactive molecules (e.g., nitric oxide, hydrogen peroxide), and biomolecules (e.g., sugars, hormones). The major challenges for microsensors are overcoming relatively low sensitivity and low signal-to-noise ratio. Modern approaches for enhancing microsensor performance focus on the incorporation of catalytic nanomaterials to increase sensitivity, reduce response time, and increase operating range. To improve signal-to-noise ratio, a non-invasive microsensor modality called self-referencing (SR) is being applied. The SR technique allows measurement of temporal and spatial transport dynamics at the cell, tissue, organ, and organismal level.

The accurate and rapid measurement of physiological O₂ transport is vital for understanding spatially and temporally dynamic metabolism and stress signalling in plant cells and tissues. Single channel luminescent O₂- quenched optrodes have been available for use in laboratory and field experiments since the early 2000’s. However, to collect the large datasets needed to understand O₂ transport in complex systems, many experiments require a multiple channel O₂ sensor system. This research reports the development of a multiplexing fiber optic O₂ microsensor system designed to conduct high temporal resolution experiments for field studies of plant physiology. The 10 channel system was demonstrated for measuring O₂ concentration in developing soybean seeds (Glycine max L. Merr.) within a climate controlled greenhouse.

The ability to conveniently and immediately test and diagnose in a diverse and rapidly changing environment is critical for field diagnostics. Smart biomedical sensors employ many different diagnostic/therapeutic methodologies; however, an ideal system would include the ability for results to be shared instantaneously with all members of the team through a software interface. We discuss our efforts towards the development and use of a robust, mobile platform (Android-based smart phone) that incorporates stable molecular recognition elements in sensor development. The inexpensive, compact, robust, archival, and portable design is ideal for rapid field diagnostics.

Graphene’s controllable optical conductivity and mechanically strong structure make it a suitable material to de- sign tunable localized surface plasmon resonance (LSPR) sensors. In this work, we theoretically and numerically demonstrate that the resonance wavelength of an LSPR sensor can be tuned to any value within a reasonably wide range of wavelengths by changing the voltage applied to graphene layer. Theoretical results reveal a higher sensitivity with respect to regular LSPR sensors.

Side effects of chemotherapy are major problems associated with current cancer treatment. An effective way to minimize these side effects and improve the efficacy of cancer treatment is to deliver drugs specifically targeted to tumors. This can be achieved by encapsulating chemotherapy drugs inside nanoparticles that aggregate in tumors due to the enhanced permeability and retention effect.

In order to monitor the delivery of nanoparticle-drug conjugates, it is important to develop systems that can image the nanoparticles. Since two-photon fluorescent probes can lead to significant reduction of background fluorescence compared to single photon fluorescent probes, two-photon fluorescent nanoparticles were developed through the miniemulsion process, using a conjugated polymer—poly [2-(3-thienyl)ethanol butoxycarbonyl-methyl urethane])—and two surfactants—sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB).

Nanoparticle size decreased as surfactant concentration increased, and particle size remained constant for surfactant concentrations above the critical micellar concentration (CMC), which was 8.2 μM for SDS and 1 μM for CTAB. The average size of the nanoparticles with surfactants at CMC was 31.67 nm for SDS nanoparticles and 25.60 nm for CTAB nanoparticles. Both nanoparticle systems exhibited strong one-photon and two-photon fluorescent signals. Fluorescence microscopy demonstrated these nanoparticles were able to penetrate rat cardiomyocytes. The results suggest these nanoparticles may potentially be used for high-contrast cell imaging.

The aim of nanodiagnostics is to identify disease at its earliest stage, particularly at the molecular level. Nanoparticlebased molecular imaging has set a unique platform for cellular tracking, targeted diagnostic studies, and imagemonitored therapy. In the preclinical setting, several modalities, such as fluorescence, positron emission tomography (PET), magnetic resonance imaging (MRI), computed tomography (CT) and ultrasound imaging are used for imaging of the cardiovascular system. Although this conventional imaging describes the extent and severity of cardiovascular diseases such as atherosclerosis or ischemia, molecular imaging is needed to identifying precursors of disease development and progression. Bringing multimodality capability to molecular imaging will harness the complimentary abilities of different techniques, thus optimizing the overall resolution and sensitivity of the resulting scans. The enhanced imaging details will permit more precise diagnosis and control of treatments.

In this paper, we present the synthesis and characterization of a dual-imaging contrast agent based on bifunctional gold nanoparticles designed for the targeting of tissue ACE (angiotensin-converting enzyme) and monitoring of cardiovascular diseases. Lisinopril (an ACE inhibitor) was selected as the targeting agent and derivatized with thioctic acid for a stronger anchoring onto gold nanoparticles. A Gd(DOTA) complex was chosen as the MRI tag. The gold core serves as the CT contrast agent. The new nanoprobes prepared not only possess the ability to target tissue ACE but also provided bimodal imaging capabilities (CT and MRI). This bimodal molecular imaging will improve the ability to accurately target diseased tissue at a very early stage, thus diagnosing and then treating patients in the most efficient way.

We studied the growth and design of solid-solution crystal of mercurous chloride (Hg₂Cl₂) and mercurous bromide (Hg₂Br₂). The lattice parameters of the mixtures obey Vagard’s law in the studied composition range. The study demonstrates that properties are very anisotropic with crystal orientation, and performance achievement requires extremely careful fabrication to utilize theoretical AO figure of merit. In addition, some parameters such as crystal growth fabrication, processing time, resolution, field of view and efficiency will be described for imagers based on novel solid solution materials. It was predicted that very similar to the mercurous chloride and mercurous bromide solid solutions also have very large anisotropy, and acousto-optic figure of merit decreases significantly as function of the crystal orientation.

Hyperspectral imaging is an emerging technology in the field of biomedical engineering which may be used as a noninvasive modality to characterize tumors. In this paper, a hyperspectral imaging system was used to characterize canine mammary tumors of unknown histopathology (pre-surgery) and correlate these results with the post-surgical histopathology results. The system consisted of a charge coupled device (CCD) camera, a liquid crystal tunable filter in the near infrared range (650-1100 nm) and a controller. Spectral signatures of malignant and benign canine mammary tumors were extracted and analyzed. The reflectance intensities of malignant tumor spectra were generally lower than benign tumor spectra over the entire wavelength range. Previous studies have shown that cancerous tissues have a higher hemoglobin and water content, and lower lipid concentration with respect to benign tissues. The decreased reflectance intensity observed for malignant tumors is likely due to the increased microvasculature and therefore higher blood content of malignant tissue relative to benign tissue. Peaks at 700, 840, 900 and 970 nm were observed in the second derivative absorption spectra, these peaks were attributed to deoxy-hemoglobin, oxy-hemoglobin, lipid and water respectively. A ‘Tissue Optical Index’ was developed that enhances contrast between malignant and benign canine tumors. This index is based on the ratio of the reflectance intensity values corresponding to the wavelengths associated with the four chromophores. Preliminary results from 22 canine mammary tumors showed that the sensitivity and specificity of the proposed method is 85.7% and 94.6% respectively. These results show promise in the non-invasive optical diagnosis of canine mammary cancer.

Medical diagnostic devices based on photoacoustics represent an emerging area with significant potential for evaluation of brain injury and chemical agent exposure, as well as detection of pandemic diseases and cancer. However, few studies have addressed photothermal safety of these devices which emit high-power laser pulses to generate rapid, selective, yet non-destructive heating of subsurface structures. Towards elucidation of laser-tissue interactions and factors of safety for photothermal injury, we have developed a three-dimensional numerical model including light propagation, heat transfer and thermal damage algorithms. Literature surveys were performed to identify appropriate optical properties and the range of device exposure levels implemented in prior in vivo studies. Initial simulations provided model validation against results from the literature. Simulations were then performed based on breast tissue with discrete blood vessels irradiated by a train of laser pulses (10 Hz) at 800 and 1064 nm. For a constant exposure level, increasing beam diameter from 0.2 to 2.0 cm led to a factor of 2.5 increase in subsurface heat generation rates. Our preliminary modeling results indicate that for a 10 second tissue exposure under standard photoacoustic imaging conditions, irradiance-based safety limits should provide a factor of safety of 6 or greater over exposure levels that induce thermal coagulation. Opticalthermal modeling represents a powerful tool for elucidating photothermal effects relevant to the safety and effectiveness of photoacoustic systems.

The emerging technique of three-dimensional (3D) printing provides a simple, fast, and flexible way to fabricate structures with arbitrary spatial features and may prove useful in the development of standardized, phantom-based performance test methods for biophotonic imaging. Acrylonitrile Butadiene Styrene (ABS) is commonly used in the printing process, given its low cost and strength. In this study, we evaluate 3D printing as an approach for fabricating biologically-relevant optical phantoms for hyperspectral reflectance imaging (HRI). The initial phase of this work involved characterization of absorption and scattering coefficients using spectrophotometry. The morphology of phantoms incorporating vessel-like channels with diameters on the order of hundreds of microns was examined by microscopy and OCT. A near-infrared absorbing dye was injected into channels located at a range of depths within the phantom and imaged with a near-infrared HRI system (650-1100 nm). ABS was found to have scattering coefficients comparable to biological tissue and low absorption throughout much of the visible and infrared range. Channels with dimensions on the order of the resolution limit of the 3D printer (~0.2 mm) exhibited pixelation effects as well as a degree of distortion along their edges. Furthermore, phantom porosity sometimes resulted in leakage from channel regions. Contrast-enhanced channel visualization with HRI was possible to a depth of nearly 1 mm – a level similar to that seen previously in biological tissue. Overall, our ABS phantoms demonstrated a high level of optical similarity to biological tissue. While limitations in printer resolution, matrix homogeneity and optical property tunability remain challenging, 3D printed phantoms have significant promise as samples for objective, quantitative evaluation of performance for biophotonic imaging modalities such as HRI.

It has been shown that using non-resonant multiphoton photoacoustic spectroscopy (NMPPAS), excised brain tumor (grade III astrocytoma) and healthy tissue can be differentiated from each other, even in neighboring biopsy samples[1, 2]. Because of this, this powerful technique offers a great deal of potential for use as a surgical guidance technique for tumor margining with up to cellular level spatial resolution[3]. NMPPAS spectra are obtained by monitoring the non-radiative relaxation pathways via ultrasonic detection, following two-photon excitation with light in the optical diagnostic window (740nm-1100nm). Based upon significant differences in the ratiometric absorption of the tissues following 970nm and 1100nm excitation, a clear classification of the tissue can be made. These differences are the result of variations in composition and oxidation state of certain endogenous biochemical species between healthy and malignant tissues. In this work, NADH, NAD⁺ and ATP were measured using NMPPAS in model gelatin tissue phantoms to begin to understand which species might be responsible for the observed spectral differences in the tissue. Each species was placed in specific pH environments to provide control over the ratio of oxidized to reduced forms of the species. Ratiometric analyses were then conducted to account for variability caused due to instrumental parameters. This paper will discuss the potential roles of each of the species for tumor determination and their contribution to the spectral signature.