R. T. Zaman, Rajaram, N., Walsh, A., Oliver, J., Rylander, H. G., Tunnell, J. W., Welch, A. J., and Mahadevan-Jansen, A., “Variation of fluorescence in tissue with temperature.,” Lasers Surg Med, vol. 43, no. 1, pp. 36-42, 2011.Abstract
BACKGROUND AND OBJECTIVE: Previous studies demonstrated a decrease in fluorescence intensity as tissue temperature increased. In vitro samples were increased from room temperature and in vivo canine liver from body temperature. This study investigated variations in fluorescence intensity with temperatures starting at 14°C and compared in vivo and in vitro results for consistency. STUDY DESIGN/MATERIAL AND METHODS: A fiber optic-based noninvasive system was used to characterize the temperature effect on tissue fluorescence in hamster dorsal skin in vivo, and in sclera and cornea of enucleated pig eyes in vitro. As tissue was allowed to progress through the temperature range of 14-42°C, the spectra of auto-fluorescence with respect to temperature was sampled every 1-2 minutes. A pulsed nitrogen laser was used to excite fluorescence through a fiber optic probe with a source-detector aperture separation of 370 µm. RESULTS: Fluorescence intensity decreased as temperature increased from 14 to 42°C in a phantom containing Rhodamine B dye. Results from both in vivo and in vitro tissue followed the same trend of decreasing intensity as tissue temperature increased from 14°C. Spectral intensity lineshape changed around 450 nm due to absorption from tissue. CONCLUSION: Cooling a tissue increased fluorescence intensity of skin in vivo, in all experiments. In vitro results were consistent with in vivo measurements.
S. F. Bish, Rajaram, N., Nichols, B., and Tunnell, J. W., “Development of a noncontact diffuse optical spectroscopy probe for measuring tissue optical properties.,” J Biomed Opt, vol. 16, no. 12, pp. 120505, 2011.Abstract
Optical reflectance probes are often used as tools to obtain optical spectra from superficial tissues and subsequently determine optical and physiological properties associated with early stage cancer. These probes, when placed directly on the tissue, are known to cause significant pressure-dependent changes in local optical properties. To address this, we fit the probe with an optical device that images the illumination and collection fibers onto the tissue surface, eliminating the influence of contact probe pressure on the sampling area. The noncontact probe addition addresses new optical conditions that may affect its performance such as tissue surface contour, and specular reflections by implementing an autofocusing mechanism and cross polarization. Extracted optical properties of tissue simulating phantoms yield errors of 3.46% in reduced scattering and 8.62% in absorbance. Autofocusing has extended the depth of field from 4 mm to throughout the 12 mm range of autofocus travel, while cross polarization has removed the incidence angle dependent specular reflection component from the collected signal.
I. Barman, Dingari, N. C., Rajaram, N., Tunnell, J. W., Dasari, R. R., and Feld, M. S., “Rapid and accurate determination of tissue optical properties using least-squares support vector machines.,” Biomed Opt Express, vol. 2, no. 3, pp. 592-9, 2011.Abstract
Diffuse reflectance spectroscopy (DRS) has been extensively applied for the characterization of biological tissue, especially for dysplasia and cancer detection, by determination of the tissue optical properties. A major challenge in performing routine clinical diagnosis lies in the extraction of the relevant parameters, especially at high absorption levels typically observed in cancerous tissue. Here, we present a new least-squares support vector machine (LS-SVM) based regression algorithm for rapid and accurate determination of the absorption and scattering properties. Using physical tissue models, we demonstrate that the proposed method can be implemented more than two orders of magnitude faster than the state-of-the-art approaches while providing better prediction accuracy. Our results show that the proposed regression method has great potential for clinical applications including in tissue scanners for cancer margin assessment, where rapid quantification of optical properties is critical to the performance.
Biophotonics Series: In vivo clinical imaging and diagnostics. New York: McGraw-Hill, 2011.
N. Rajaram, Gopal, A., Zhang, X., and Tunnell, J. W., “Experimental validation of the effects of microvasculature pigment packaging on in vivo diffuse reflectance spectroscopy.,” Lasers Surg Med, vol. 42, no. 7, pp. 680-8, 2010.Abstract
BACKGROUND: Diffuse reflectance spectroscopy (DRS) uses the steady-state diffuse reflectance measured from the tissue surface to determine absorption and scattering properties of sampled tissue. Many inverse models used to determine absorber properties have assumed a homogeneous distribution of blood. However, blood in tissue is confined to blood vessels that occupy a small fraction of the overall volume. This simplified assumption can lead to large errors when measuring optical properties. The objective of this study was to examine the effect of confining absorbers to small volumes, such as the microvasculature, on in vivo DRS. STUDY DESIGN: We fabricated multi-layer microfluidic devices to mimic blood vessels with a size similar to skin microvasculature. We studied the effect of varying channel size (diameter = 22 and 44 microm) and absorber concentration (10-80% food color dye in water) on diffuse reflectance measurements. We also examined the in vivo reflectance from normal skin and non-melanoma skin cancer on 14 patients. RESULTS: Our results demonstrate that both absorption coefficient and vessel diameter affect the diffuse reflectance spectra. An empirically calculated packaging correction factor based on our experiments shows good agreement with previous theoretical derivations of the same factor. In vivo measurements on normal skin and basal cell carcinoma show that incorporating a correction factor greatly improves the fit of the inverse model to the spectra. In addition, there were statistically significant differences in measured mean vessel diameter and blood volume fraction between normal skin and basal cell carcinoma. CONCLUSION: We have demonstrated experimentally the effect of pigment packaging in blood vessels over a physiologically relevant range of blood vessel size and absorption. The correction factors implemented to account for the packaging effect could potentially be used as diagnostic parameters for diagnosing skin cancers.
J. Park, Estrada, A., Schwartz, J. A., Diagaradjane, P., Krishnan, S., Dunn, A. K., and Tunnell, J. W., “Intra-organ Biodistribution of Gold Nanoparticles Using Intrinsic Two-photon Induced Photoluminescence.,” Lasers Surg Med, vol. 42, no. 7, pp. 630-639, 2010.Abstract
BACKGROUND AND OBJECTIVES: Gold nanoparticles (GNPs) such as gold nanoshells (GNSs) and gold nanorods (GNRs) have been explored in a number of in vitro and in vivo studies as imaging contrast and cancer therapy agents due to their highly desirable spectral and molecular properties. While the organ-level biodistribution of these particles has been reported previously, little is known about the cellular level or intra-organ biodistribution. The objective of this study was to demonstrate the use of intrinsic two-photon induced photoluminescence (TPIP) to study the cellular level biodistribution of GNPs. STUDY DESIGN/MATERIALS AND METHODS: Tumor xenografts were created in twenty-seven male nude mice (Swiss nu/nu) using HCT 116 cells (CCL-247, ATCC, human colorectal cancer cell line). GNSs and GNRs were systemically injected 24 hr. prior to tumor harvesting. A skin flap with the tumor was excised and sectioned as 8 μm thick tissues for imaging GNPs under a custom-built multiphoton microscope. For multiplexed imaging, nuclei, cytoplasm, and blood vessels were demonstrated by hematoxylin and eosin (H&E) staining, YOYO-1 iodide staining and CD31-immunofluorescence staining. RESULTS: Distribution features of GNPs at the tumor site were determined from TPIP images. GNSs and GNRs had a heterogeneous distribution with higher accumulation at the tumor cortex than tumor core. GNPs were also observed in unique patterns surrounding the perivascular region. While most GNSs were confined at the distance of approximately 400 μm inside the tumor edge, GNRs were shown up to 1.5 mm penetration inside the edge. CONCLUSIONS: We have demonstrated the use of TPIP imaging in a multiplexed fashion to image both GNPs and nuclei, cytoplasm, or vasculature simultaneously. We also confirmed that TPIP imaging enabled visualization of GNP distribution patterns within the tumor and other critical organs. These results suggest that direct luminescence-based imaging of metal nanoparticles holds a valuable and promising position in understanding the accumulation kinetics of GNPs. In addition, these techniques will be increasingly important as the use of these particles progress to human clinical trials where standard histopathology techniques are used to analyze their effects.
Y. Wang, Bish, S., Tunnell, J. W., and Zhang, X., “MEMS scanner enabled real-time depth sensitive hyperspectral imaging of biological tissue.,” Opt Express, vol. 18, no. 23, pp. 24101-8, 2010.Abstract
We demonstrate a hyperspectral and depth sensitive diffuse optical imaging microsystem, where fast scanning is provided by a CMOS compatible 2-axis MEMS mirror. By using lissajous scanning patterns, large field-of-view (FOV) of 1.2 cmx1.2 cm images with lateral resolution of 100 µm can be taken at 1.3 frames-per-second (fps). Hyperspectral and depth-sensitive images were acquired on tissue simulating phantom samples containing quantum dots (QDs) patterned at various depths in Polydimethylsiloxane (PDMS). Device performance delivers 6 nm spectral resolution and 0.43 wavelengths per second acquisition speed. A sample of porcine epithelium with subcutaneously placed QDs was also imaged. Images of the biological sample were processed by spectral unmixing in order to qualitatively separate chromophores in the final images and demonstrate spectral performance of the imaging system.
T. A. Erickson, Mazhar, A., Cuccia, D., Durkin, A. J., and Tunnell, J. W., “Lookup-table method for imaging optical properties with structured illumination beyond the diffusion theory regime.,” J Biomed Opt, vol. 15, no. 3, pp. 036013, 2010.Abstract
Sinusoidally structured illumination is used in concert with a phantom-based lookup-table (LUT) to map wide-field optical properties in turbid media with reduced albedos as low as 0.44. A key advantage of the lookup-table approach is the ability to measure the absorption (mu(a)) and reduced scattering coefficients (mu(s) (')) over a much broader range of values than permitted by current diffusion theory methods. Through calibration with a single reflectance standard, the LUT can extract mu(s) (') from 0.8 to 2.4 mm(-1) with an average root-mean-square (rms) error of 7% and extract mu(a) from 0 to 1.0 mm(-1) with an average rms error of 6%. The LUT is based solely on measurements of two parameters, reflectance R and modulation M at an illumination period of 10 mm. A single set of three phase-shifted images is sufficient to measure both M and R, which are then used to generate maps of absorption and scattering by referencing the LUT. We establish empirically that each pair (M,R) maps uniquely to only one pair of (micro(s) ('),micro(a)) and report that the phase function (i.e., size) of the scatterers can influence the accuracy of optical property extraction.
N. Rajaram, Aramil, T. J., Lee, K., Reichenberg, J. S., Nguyen, T. H., and Tunnell, J. W., “Design and validation of a clinical instrument for spectral diagnosis of cutaneous malignancy.,” Appl Opt, vol. 49, no. 2, pp. 142-52, 2010.Abstract
We report a probe-based portable and clinically compatible instrument for the spectral diagnosis of melanoma and nonmelanoma skin cancers. The instrument combines two modalities--diffuse reflectance and intrinsic fluorescence spectroscopy--to provide complementary information regarding tissue morphology, function, and biochemical composition. The instrument provides a good signal-to-noise ratio for the collected reflectance and laser-induced fluorescence spectra. Validation experiments on tissue phantoms over a physiologically relevant range of albedos (0.35-0.99) demonstrate an accuracy of close to 10% in determining scattering, absorption and fluorescence characteristics. We also demonstrate the ability of our instrument to collect in vivo diffuse reflectance and fluorescence measurements from clinically normal skin, dysplastic nevus, and malignant nonmelanoma skin cancer.
N. Rajaram, Reichenberg, J. S., Migden, M. R., Nguyen, T. H., and Tunnell, J. W., “Pilot clinical study for quantitative spectral diagnosis of non-melanoma skin cancer.,” Lasers Surg Med, vol. 42, no. 10, pp. 716-27, 2010.Abstract
BACKGROUND: Several research groups have demonstrated the non-invasive diagnostic potential of diffuse optical spectroscopy (DOS) and laser-induced fluorescence (LIF) techniques for early cancer detection. By combining both modalities, one can simultaneously measure quantitative parameters related to the morphology, function and biochemical composition of tissue and use them to diagnose malignancy. The objective of this study was to use a quantitative reflectance/fluorescence spectroscopic technique to determine the optical properties of normal skin and non-melanoma skin cancers and the ability to accurately classify them. An additional goal was to determine the ability of the technique to differentiate non-melanoma skin cancers from normal skin. STUDY DESIGN: The study comprised 48 lesions measured from 40 patients scheduled for a biopsy of suspected non-melanoma skin cancers. White light reflectance and laser-induced fluorescence spectra (wavelength range = 350-700 nm) were collected from each suspected lesion and adjacent clinically normal skin using a custom-built, optical fiber-based clinical instrument. After measurement, the skin sites were biopsied and categorized according to histopathology. Using a quantitative model, we extracted various optical parameters from the measured spectra that could be correlated to the physiological state of tissue. RESULTS: Scattering from cancerous lesions was significantly lower than normal skin for every lesion group, whereas absorption parameters were significantly higher. Using numerical cut-offs for our optical parameters, our clinical instrument could classify basal cell carcinomas with a sensitivity and specificity of 94% and 89%, respectively. Similarly, the instrument classified actinic keratoses and squamous cell carcinomas with a sensitivity of 100% and specificity of 50%. CONCLUSION: The measured optical properties and fluorophore contributions of normal skin and non-melanoma skin cancers are significantly different from each other and correlate well with tissue pathology. A diagnostic algorithm that combines these extracted properties holds promise for the potential non-invasive diagnosis of skin cancer.
C. Yan, Pattani, V., Tunnell, J. W., and Ren, P., “Temperature-induced unfolding of epidermal growth factor (EGF): insight from molecular dynamics simulation.,” J Mol Graph Model, vol. 29, no. 1, pp. 2-12, 2010.Abstract
Thermal disruption of protein structure and function is a potentially powerful therapeutic vehicle. With the emerging nanoparticle-targeting and femtosecond laser technology, it is possible to deliver heating locally to specific molecules. It is therefore important to understand how fast a protein can unfold or lose its function at high temperatures, such as near the water boiling point. In this study, the thermal damage of EGF was investigated by combining the replica exchange (136 replicas) and conventional molecular dynamics simulations. The REMD simulation was employed to rigorously explore the free-energy landscape of EGF unfolding. Interestingly, besides the native and unfolded states, we also observed a distinct molten globule (MG) state that retained substantial amount of native contacts. Based on the understanding that which the unfolding of EGF is a three-state process, we have examined the unfolding kinetics of EGF (N-->MG and MG-->D) with multiple 20-ns conventional MD simulations. The Arrhenius prefactors and activation energy barriers determined from the simulation are within the range of previously studied proteins. In contrast to the thermal damage of cells and tissues which take place on the time scale of seconds to hours at relatively low temperatures, the denaturation of proteins occur in nanoseconds when the temperature of heat bath approaches the boiling point.
C. M. Hessel, Rasch, M., Hueso, J. L., Goodfellow, B., Akhavan, V. A., Puvanakrishnan, P., Tunnell, J. W., and Korgel, B. A., “Alkyl Passivation and Amphiphilic Polymer Coating of Silicon Nanocrystals for Medical Diagnostic Imaging,” Small, vol. 6, pp. 2026–2034, 2010.
C. Lau, Sćepanović, O., Mirkovic, J., McGee, S., Yu, C. - C., Fulghum, S., Wallace, M., Tunnell, J., Bechtel, K., and Feld, M., “Re-evaluation of model-based light-scattering spectroscopy for tissue spectroscopy.,” J Biomed Opt, vol. 14, no. 2, pp. 024031, 2009.Abstract
Model-based light scattering spectroscopy (LSS) seemed a promising technique for in-vivo diagnosis of dysplasia in multiple organs. In the studies, the residual spectrum, the difference between the observed and modeled diffuse reflectance spectra, was attributed to single elastic light scattering from epithelial nuclei, and diagnostic information due to nuclear changes was extracted from it. We show that this picture is incorrect. The actual single scattering signal arising from epithelial nuclei is much smaller than the previously computed residual spectrum, and does not have the wavelength dependence characteristic of Mie scattering. Rather, the residual spectrum largely arises from assuming a uniform hemoglobin distribution. In fact, hemoglobin is packaged in blood vessels, which alters the reflectance. When we include vessel packaging, which accounts for an inhomogeneous hemoglobin distribution, in the diffuse reflectance model, the reflectance is modeled more accurately, greatly reducing the amplitude of the residual spectrum. These findings are verified via numerical estimates based on light propagation and Mie theory, tissue phantom experiments, and analysis of published data measured from Barrett's esophagus. In future studies, vessel packaging should be included in the model of diffuse reflectance and use of model-based LSS should be discontinued.
P. Puvanakrishnan, Park, J., Diagaradjane, P., Schwartz, J. A., Coleman, C. L., Gill-Sharp, K. L., Sang, K. L., Payne, D. J., Krishnan, S., and Tunnell, J. W., “Near-infrared narrow-band imaging of gold/silica nanoshells in tumors.,” J Biomed Opt, vol. 14, no. 2, pp. 024044, 2009.Abstract
Gold nanoshells (GNS) are a new class of nanoparticles that can be optically tuned to scatter or absorb light from the near-ultraviolet to near-infrared (NIR) region by varying the core (dielectric silica)/shell (gold) ratio. In addition to spectral tunability, GNS are inert and bioconjugatable, making them potential labels for in vivo imaging and therapy of tumors. We report the use of GNS as exogenous contrast agents for enhanced visualization of tumors using narrow-band imaging (NBI). NBI takes advantage of the strong NIR absorption of GNS to distinguish between blood and nanoshells in the tumor by imaging in narrow wavelength bands in the visible and NIR, respectively. Using tissue-simulating phantoms, we determined the optimum wavelengths to enhance contrast between blood and GNS. We then used the optimum wavelengths for ex vivo imaging of tumors extracted from human colon cancer xenograft bearing mice injected with GNS. Systemically delivered GNS accumulated passively in tumor xenografts by the enhanced permeability and retention (EPR) effect. Ex vivo NBI of tumor xenografts demonstrated heterogeneous distribution of GNS with a clear distinction from the tumor vasculature. The results of this study demonstrate the feasibility of using GNS as contrast agents to visualize tumors using NBI.
T. Erickson and Tunnell, J. W., “Gold Nanoshells in Biomedical Applications,” in Nanomaterials for the Life Sciences: Mixed metal nanomaterials, vol. 3, 2009, pp. 1–44.
N. Rajaram, Nguyen, T. H., and Tunnell, J. W., “Lookup table-based inverse model for determining optical properties of turbid media.,” J Biomed Opt, vol. 13, no. 5, pp. 050501, 2008.Abstract
We present a lookup table (LUT)-based inverse model for determining the optical properties of turbid media from steady-state diffuse reflectance spectra that is valid for fiber-based probe geometries with close source-detector separations and tissue with low albedo. The lookup table is based solely on experimental measurements of calibration standards. We used tissue-simulating phantoms to validate the accuracy of the LUT inverse model. Our results show excellent agreement between the expected and extracted values of the optical parameters. In addition, the LUT represents a significant improvement in accuracy at short source-detector separations (300 microm) and low albedo (approximately 0.35). We also present in vivo data from clinically normal and malignant nonmelanoma skin cancers fit to the LUT-based model.
P. Diagaradjane, Shetty, A., Wang, J. C., Elliott, A. M., Schwartz, J., Shentu, S., Park, H. C., Deorukhkar, A., Stafford, J. R., Cho, S. H., Tunnell, J. W., Hazle, J. D., and Krishnan, S., “Modulation of in vivo tumor radiation response via gold nanoshell-mediated vascular-focused hyperthermia: characterizing an integrated antihypoxic and localized vascular disrupting targeting strategy.,” Nano Lett, vol. 8, no. 5, pp. 1492-500, 2008.Abstract
We report noninvasive modulation of in vivo tumor radiation response using gold nanoshells. Mild-temperature hyperthermia generated by near-infrared illumination of gold nanoshell-laden tumors, noninvasively quantified by magnetic resonance temperature imaging, causes an early increase in tumor perfusion that reduces the hypoxic fraction of tumors. A subsequent radiation dose induces vascular disruption with extensive tumor necrosis. Gold nanoshells sequestered in the perivascular space mediate these two tumor vasculature-focused effects to improve radiation response of tumors. This novel integrated antihypoxic and localized vascular disrupting therapy can potentially be combined with other conventional antitumor therapies.
J. Park, Estrada, A., Sharp, K., Sang, K., Schwartz, J. A., Smith, D. K., Coleman, C., Payne, J. D., Korgel, B. A., Dunn, A. K., and Tunnell, J. W., “Two-photon-induced photoluminescence imaging of tumors using near-infrared excited gold nanoshells.,” Opt Express, vol. 16, no. 3, pp. 1590-9, 2008.Abstract
Gold nanoshells (dielectric silica core/gold shell) are a novel class of hybrid metal nanoparticles whose unique optical properties have spawned new applications including more sensitive molecular assays and cancer therapy. We report a new photo-physical property of nanoshells (NS) whereby these particles glow brightly when excited by near-infrared light. We characterized the luminescence brightness of NS, comparing to that of gold nanorods (NR) and fluorescent beads (FB). We find that NS are as bright as NR and 140 times brighter than FB. To demonstrate the potential application of this bright two-photon-induced photoluminescence (TPIP) signal for biological imaging, we imaged the 3D distribution of gold nanoshells targeted to murine tumors.
R. T. Zaman, Diagaradjane, P., Wang, J., Swartz, J., Rajaram, N., Gill-Sharp, K., Cho, S., Rylander, H. G., Payne, D. J., Krishnan, S., and Tunnell, J. W., “In vivo detection of gold nanoshells in tumors using diffuse optical spectroscopy,” IEEE J Sel Top Quant Elec, vol. 13, pp. 1715–1720, 2007.
C. - C. Yu, Lau, C., Tunnell, J. W., Hunter, M., Kalashnikov, M., Fang-Yen, C., Fulghum, S. F., Badizadegan, K., Dasari, R. R., and Feld, M. S., “Assessing epithelial cell nuclear morphology by using azimuthal light scattering spectroscopy.,” Opt Lett, vol. 31, no. 21, pp. 3119-21, 2006.Abstract
We describe azimuthal light scattering spectroscopy (phi/LSS), a novel technique for assessing epithelial-cell nuclear morphology. The difference between the spectra measured at azimuthal angles phi = 0 degrees and phi = 90 degrees preferentially isolates the single backscattering contribution due to large (approximately 10 microm) structures such as epithelial cell nuclei by discriminating against scattering from smaller organelles and diffusive background. We demonstrate the feasibility of using phi/LSS for cancer detection by showing that spectra from cancerous colon tissue exhibit significantly greater azimuthal asymmetry than spectra from normal colonic tissues.