R. Hennessy, Goth, W., Sharma, M., Markey, M. K., and Tunnell, J. W., “Effect of probe geometry and optical properties on the sampling depth for diffuse reflectance spectroscopy,” Journal of Biomedical Optics, vol. 19, pp. 107002, 2014. Publisher's VersionAbstract
Abstract.  The sampling depth of light for diffuse reflectance spectroscopy is analyzed both experimentally and computationally. A Monte Carlo (MC) model was used to investigate the effect of optical properties and probe geometry on sampling depth. MC model estimates of sampling depth show an excellent agreement with experimental measurements over a wide range of optical properties and probe geometries. The MC data are used to define a mathematical expression for sampling depth that is expressed in terms of optical properties and probe geometry parameters.
B. Yang and Tunnell, J. W., “Real-time absorption reduced surface fluorescence imaging,” Journal of Biomedical Optics, vol. 19, pp. 090505, 2014. Publisher's VersionAbstract
Abstract.  We introduce a technique that limits absorption effects in fluorescence imaging and does not require extensive imaging processing, thus allowing for video rate imaging. The absorption minimization is achieved using spatial frequency domain imaging at a single high spatial frequency with standard three-phase demodulation. At a spatial frequency f=0.5  mm−1, we demonstrated in both in-vitro phantoms and ex-vivo tissue that the absorption can be significantly reduced. In the real-time implementation, we achieved a video rate of 19  frames/s. This technique has potential in cancer visualization and tumor margin detection.
M. Sharma, Marple, E., Reichenberg, J., and Tunnell, J. W., “Design and characterization of a novel multimodal fiber-optic probe and spectroscopy system for skin cancer applications,” Review of Scientific Instruments, vol. 85, pp. -, 2014. Publisher's Version
R. Hennessy, Lim, S. L., Markey, M. K., and Tunnell, J. W., “Monte Carlo lookup table-based inverse model for extracting optical properties from tissue-simulating phantoms using diffuse reflectance spectroscopy.,” J Biomed Opt, vol. 18, no. 3, pp. 037003, 2013.Abstract
We present a Monte Carlo lookup table (MCLUT)-based inverse model for extracting optical properties from tissue-simulating phantoms. This model is valid for close source-detector separation and highly absorbing tissues. The MCLUT is based entirely on Monte Carlo simulation, which was implemented using a graphics processing unit. We used tissue-simulating phantoms to determine the accuracy of the MCLUT inverse model. Our results show strong agreement between extracted and expected optical properties, with errors rate of 1.74% for extracted reduced scattering values, 0.74% for extracted absorption values, and 2.42% for extracted hemoglobin concentration values.
M. Sharma, Hennessy, R., Markey, M. K., and Tunnell, J. W., “Verification of a two-layer inverse Monte Carlo absorption model using multiple source-detector separation diffuse reflectance spectroscopy.,” Biomed Opt Express, vol. 5, no. 1, pp. 40-53, 2013.Abstract
A two-layer Monte Carlo lookup table-based inverse model is validated with two-layered phantoms across physiologically relevant optical property ranges. Reflectance data for source-detector separations of 370 μm and 740 μm were collected from these two-layered phantoms and top layer thickness, reduced scattering coefficient and the top and bottom layer absorption coefficients were extracted using the inverse model and compared to the known values. The results of the phantom verification show that this method is able to accurately extract top layer thickness and scattering when the top layer thickness ranges from 0 to 550 μm. In this range, top layer thicknesses were measured with an average error of 10% and the reduced scattering coefficient was measured with an average error of 15%. The accuracy of top and bottom layer absorption coefficient measurements was found to be highly dependent on top layer thickness, which agrees with physical expectation; however, within appropriate thickness ranges, the error for absorption properties varies from 12-25%.
B. Yang, Sharma, M., and Tunnell, J. W., “Attenuation-corrected fluorescence extraction for image-guided surgery in spatial frequency domain.,” J Biomed Opt, vol. 18, no. 8, pp. 80503, 2013.Abstract
A new approach to retrieve the attenuation-corrected fluorescence using spatial frequency-domain imaging is demonstrated. Both in vitro and ex vivo experiments showed the technique can compensate for the fluorescence attenuation from tissue absorption and scattering. This approach has potential in molecular image-guided surgery.
B. Yang, Sharma, M., Wang, Y., and Tunnell, J. W., “Attenuation corrected fluorescence extraction using spatial frequency domain imaging system,” Proc. SPIE, vol. 8618. pp. 861809-861809-5, 2013. Publisher's VersionAbstract
In this paper, we present a novel approach to retrieve attenuation corrected fluorescence (ACF) in the image field. This approach can be applied to improve tumor identification for both diagnosis and treatment purpose. Furthermore, this approach will facilitate the development of fluorescence image-guided surgery.
V. P. Pattani and Tunnell, J. W., “Nanoparticle-mediated photothermal therapy: a comparative study of heating for different particle types.,” Lasers Surg Med, vol. 44, no. 8, pp. 675-84, 2012.Abstract
INTRODUCTION: Near-infrared (NIR) absorbing plasmonic nanoparticles enhance photothermal therapy of tumors. In this procedure, systemically delivered gold nanoparticles preferentially accumulate at the tumor site and when irradiated using laser light, produce localized heat sufficient to damage tumor cells. Gold nanoshells and nanorods have been widely studied for this purpose, and while both exhibit strong NIR absorption, their overall absorption and scattering properties differ widely due to their geometry. In this paper, we compared the photothermal response of both nanoparticle types including the heat generation and photothermal efficiency. METHODS: Tissue simulating phantoms, with varying concentrations of gold nanoparticles, were irradiated with a near-infrared diode laser while concurrently monitoring the surface temperature with an infrared camera. We calculated nanoshell and nanorod optical properties using the Mie solution and the discrete dipole approximation, respectively. In addition, we measured the heat generation of nanoshells and nanorods at the same optical density to determine the photothermal transduction efficiency for both nanoparticle types. RESULTS: We found that the gold nanoshells produced more heat than gold nanorods at equivalent number densities (# of nanoparticles/ml), whereas the nanorods generated more heat than nanoshells at equivalent extinction values at the irradiance wavelength. To reach an equivalent heat generation, we found that it was necessary to have ∼36× more nanorods than nanoshells. However, the gold nanorods were found to have two times the photothermal transduction efficiency than the gold nanoshells. CONCLUSION: For the nanoparticles tested, the nanoshells generated more heat, per nanoparticle, than nanorods, primarily due to their overall larger geometric cross-section. Conversely, we found that the gold nanorods had a higher photothermal efficiency than the gold nanoshells. In conclusion, the ideal choice of plasmonic nanoparticle requires not only per particle efficiency, but also the in vivo particle targeting ability under study.
B. S. Nichols, Rajaram, N., and Tunnell, J. W., “Performance of a lookup table-based approach for measuring tissue optical properties with diffuse optical spectroscopy.,” J Biomed Opt, vol. 17, no. 5, pp. 057001, 2012.Abstract
Diffuse optical spectroscopy (DOS) provides a powerful tool for fast and noninvasive disease diagnosis. The ability to leverage DOS to accurately quantify tissue optical parameters hinges on the model used to estimate light-tissue interaction. We describe the accuracy of a lookup table (LUT)-based inverse model for measuring optical properties under different conditions relevant to biological tissue. The LUT is a matrix of reflectance values acquired experimentally from calibration standards of varying scattering and absorption properties. Because it is based on experimental values, the LUT inherently accounts for system response and probe geometry. We tested our approach in tissue phantoms containing multiple absorbers, different sizes of scatterers, and varying oxygen saturation of hemoglobin. The LUT-based model was able to extract scattering and absorption properties under most conditions with errors of less than 5 percent. We demonstrate the validity of the lookup table over a range of source-detector separations from 0.25 to 1.48 mm. Finally, we describe the rapid fabrication of a lookup table using only six calibration standards. This optimized LUT was able to extract scattering and absorption properties with average RMS errors of 2.5 and 4 percent, respectively.
R. T. Zaman, Gopal, A., Starr, K., Zhang, X., Thomsen, S., Tunnell, J. W., Welch, A. J., and Rylander, H. G., “Micro-patterned drug delivery device for light-activated drug release.,” Lasers Surg Med, vol. 44, no. 1, pp. 30-48, 2012.Abstract
BACKGROUND: The primary goal of this study was the fabrication, long-term stability, and measured release of a marker dye from a micro-patterned drug delivery device using (i) mechanical puncture and (ii) photodisruption with an ophthalmic Nd:YAG laser. MATERIALS AND METHODS: A drug delivery device was made from a transparent bio-compatible polymer. The device consisted of two 2.6 mm diameter reservoirs containing 10% Na fluorescein dye. The device was implanted in the rabbit's eye (n = 2) with the cap of the device facing toward the exterior of the eye. Once the animals recovered from the implant surgery, 100% anhydrous glycerol was topically applied to the eye at the implantation site to decrease light scattering in the conjunctiva and sclera. The dye was released from one of the reservoir either using a 28 G ½ needle or an ophthalmic Q-switched Nd:YAG laser. A fluorescence spectrophotometer (FS) with fiber optic probe was used to measure the half-life of the dye in the eye. Measurements of fluorescence intensity were collected until the measurements return to baseline and histology was done on the tissue surrounded the device. RESULTS: None of the devices leaked of 10% Na fluorescein dye after implant. The ablation threshold of the drug delivery device was between 6 and 10 mJ to create 100-500 µm holes. The half-life measurement of the dye was found to be 13 days at the vitreous chamber after measuring the fluorescence intensity through the dilated cornea. Histology study showed minimal immune and foreign body response such as mild inflammation. CONCLUSION: This study established that the drug delivery device seemed to elicit minimal inflammatory response and retained its fluidic content until it was released with relatively longer retention time (half-life). Thus, similar device could be used for controlled release of drugs for certain ocular diseases.
W. C. Vogt, Izquierdo-Román, A., Nichols, B., Lim, L., Tunnell, J. W., and Rylander, C. G., “Effects of mechanical indentation on diffuse reflectance spectra, light transmission, and intrinsic optical properties in ex vivo porcine skin.,” Lasers Surg Med, vol. 44, no. 4, pp. 303-9, 2012.Abstract
BACKGROUND AND OBJECTIVES: Mechanical indentation has been shown to increase light transmission through turbid tissue. In this study, we investigated the effects of localized indentation on the optical properties of ex vivo porcine skin specimens by dynamically monitoring diffuse reflectance spectra, light transmission, and applied load while controlling tissue thickness. STUDY DESIGN/METHODS: A custom-built diffuse reflectance spectroscopy (DRS) system was used to capture diffuse reflectance spectra from tissue specimens undergoing indentation. The DRS probe was designed to perform both optical sensing and tissue indentation. A mechanical load frame was used to dynamically control probe displacement and resultant specimen thickness change while recording applied load. Diffuse reflectance spectra, as well as light transmission at 630 nm, were recorded during stress relaxation tests where tissue specimens were displaced to and held at a final thickness. Tissue optical properties were extracted from reflectance spectra using a previously established look-up table (LUT) approach. RESULTS: Indentation increased light transmission through tissue during linear displacement, and continued to increase transmission during subsequent stress relaxation at constant tissue thickness. The magnitude of relative transmission increases was shown to be a function of bulk tissue compressive strain (relative thickness change). Reduced scattering coefficients calculated from the LUT at 630 nm decreased during stress relaxation, with the relative decrease in scattering also depending strongly on tissue compressive strain. Reduced scattering coefficients decreased by 12.0 ± 4.7% at 0.44 ± 0.022 compressive strain, and reduced by 35.6 ± 1.3% at 0.71 ± 0.01 compressive strain. CONCLUSION: DRS can be used to capture transient changes in intrinsic tissue optical properties during mechanical loading. Mechanical indentation modifies tissue optical properties and may be harnessed as a minimally-invasive optical clearing technique to improve optical diagnostics and therapeutics.
P. Puvanakrishnan, Diagaradjane, P., Kazmi, S. S. M., Dunn, A. K., Krishnan, S., and Tunnell, J. W., “Narrow band imaging of squamous cell carcinoma tumors using topically delivered anti-EGFR antibody conjugated gold nanorods.,” Lasers Surg Med, vol. 44, no. 4, pp. 310-7, 2012.Abstract
BACKGROUND: Nanoparticles have recently gained interest as exogenous contrast agents in a variety of biomedical applications related to cancer detection and treatment. The objective of this study was to determine the potential of topically administered antibody conjugated gold nanorods (GNRs) for imaging squamous cell carcinomas (SCCs) of the skin using near-infrared narrowband imaging (NBI). Near-infrared (NIR) NBI images narrow wavelength bands to enhance contrast from plasmonic particles in a wide field portable and noncontact device that is clinically compatible for real-time tumor imaging and tumor margin demarcation. STUDY DESIGN: We conjugated GNRs to Cetuximab, a clinically approved humanized antibody that targets the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of many tumor cells, especially SCCs. We excised subcutaneous xenografts of SCCs (A431) from Swiss nu/nu mice and divided the tumors into two groups: (1) the targeted group (Cetuximab conjugated GNRs) and (2) the control group (polyethylene glycol-conjugated GNRs). After topical application of particles and incubation for 30 minutes, the tumors were washed and imaged using NBI. In addition, we performed two-photon imaging to quantify the binding of EGFR targeted GNRs in tumors and their depth profile. RESULTS: The NBI images showed a visual increase in contrast from tumors after topical administration of targeted GNR. Targeted GNR tumors showed increased contrast compared to tumors administered with the control GNR. There was a statistically significant increase in mean pixel intensity (∼2.5×) from targeted GNR tumors (n = 6). Two-photon microscopy images of targeted GNRs confirmed their binding affinity to the EGF receptors over expressed in the A431 tumors. CONCLUSION: We have demonstrated that a topical application of gold nanorods targeted specifically to tumor growth factor receptors results in a significantly higher image contrast compared to nontargeted gold nanorods. These results demonstrate the feasibility of near-infrared NBI to image and demarcate tumor margins during surgical resection using topical administration of targeted GNR.
P. Puvanakrishnan, Park, J., Chatterjee, D., Krishnan, S., and Tunnell, J. W., “In vivo tumor targeting of gold nanoparticles: effect of particle type and dosing strategy.,” Int J Nanomedicine, vol. 7, pp. 1251-8, 2012.Abstract
Gold nanoparticles (GNPs) have gained significant interest as nanovectors for combined imaging and photothermal therapy of tumors. Delivered systemically, GNPs preferentially accumulate at the tumor site via the enhanced permeability and retention effect, and when irradiated with near infrared light, produce sufficient heat to treat tumor tissue. The efficacy of this process strongly depends on the targeting ability of the GNPs, which is a function of the particle's geometric properties (eg, size) and dosing strategy (eg, number and amount of injections). The purpose of this study was to investigate the effect of GNP type and dosing strategy on in vivo tumor targeting. Specifically, we investigated the in vivo tumor-targeting efficiency of pegylated gold nanoshells (GNSs) and gold nanorods (GNRs) for single and multiple dosing. We used Swiss nu/nu mice with a subcutaneous tumor xenograft model that received intravenous administration for a single and multiple doses of GNS and GNR. We performed neutron activation analysis to quantify the gold present in the tumor and liver. We performed histology to determine if there was acute toxicity as a result of multiple dosing. Neutron activation analysis results showed that the smaller GNRs accumulated in higher concentrations in the tumor compared to the larger GNSs. We observed a significant increase in GNS and GNR accumulation in the liver for higher doses. However, multiple doses increased targeting efficiency with minimal effect beyond three doses of GNPs. These results suggest a significant effect of particle type and multiple doses on increasing particle accumulation and on tumor targeting ability.
R. Hennessy, Bish, S., Tunnell, J. W., and Markey, M. K., “Segmentation of diffuse reflectance hyperspectral datasets with noise for detection of Melanoma.,” Conf Proc IEEE Eng Med Biol Soc, vol. 2012, pp. 1482-5, 2012.Abstract
We present a segmentation algorithm that allows optical properties to be extracted from diffuse reflectance hyperspectral datasets with a speedup of three orders of magnitude when compared to current methods. Such data could be used for the detection of melanoma. The algorithm first performs dimensionality reduction using principal component analysis, and then the image is segmented using k-means clustering. The mean spectrum from each cluster is then calculated and can be used to extract chemical information. By reducing the number of spectra to be analyzed, extraction of physiological information can be achieved three orders of magnitude faster than methods requiring the analysis of every spectrum in the hyperspectral dataset. The effect of noise on the ability of the algorithm to accurately segment images was tested using digital phantoms, for which the noise level was under the control of the investigators. The analysis showed a linear relationship between the level of noise and the smallest difference in scattering that the algorithm was able to accurately detect and segment. This finding can be used to determine the maximum amount of noise in the imaging system that will still allow detection of the difference in optical properties between non-melanoma and melanoma.
S. Marek, Liechty, W., and Tunnell, J. W., “Controlled Drug Delivery from Alginate Spheres in Design-Based Learning Course,” 2012.
Y. Wang, Bish, S., Tunnell, J. W., and Zhang, X. J., “MEMS Scanner Based Handheld Fluorescent Hyperspectral Imaging System,” Sensors and Actuators: A Physical , vol. 188, pp. 450-455, 2012.
V. Pattani and Tunnell, J. W., “Optical imaging of plasmonic nanoparticles”, 2012.
C. M. Hessel, Pattani, V. P., Rasch, M., Panthani, M. G., Koo, B., Tunnell, J. W., and Korgel, B. A., “Copper selenide nanocrystals for photothermal therapy.,” Nano Lett, vol. 11, no. 6, pp. 2560-6, 2011.Abstract
Ligand-stabilized copper selenide (Cu(2-x)Se) nanocrystals, approximately 16 nm in diameter, were synthesized by a colloidal hot injection method and coated with amphiphilic polymer. The nanocrystals readily disperse in water and exhibit strong near-infrared (NIR) optical absorption with a high molar extinction coefficient of 7.7 × 10(7) cm(-1) M(-1) at 980 nm. When excited with 800 nm light, the Cu(2-x)Se nanocrystals produce significant photothermal heating with a photothermal transduction efficiency of 22%, comparable to nanorods and nanoshells of gold (Au). In vitro photothermal heating of Cu(2-x)Se nanocrystals in the presence of human colorectal cancer cell (HCT-116) led to cell destruction after 5 min of laser irradiation at 33 W/cm(2), demonstrating the viabilitiy of Cu(2-x)Se nanocrystals for photothermal therapy applications.
R. T. Zaman, Rajaram, N., Nichols, B. S., Rylander, H. G., Wang, T., Tunnell, J. W., and Welch, A. J., “Changes in morphology and optical properties of sclera and choroidal layers due to hyperosmotic agent.,” J Biomed Opt, vol. 16, no. 7, pp. 077008, 2011.Abstract
Light scattering in the normally white sclera prevents diagnostic imaging or delivery of a focused laser beam to a target in the underlying choroid layer. In this study, we examine optical clearing of the sclera and changes in blood flow resulting from the application of glycerol to the sclera of rabbits. Recovery dynamics are monitored after the application of saline. The speed of clearing for injection delivery is compared to the direct application of glycerol through an incision in the conjunctiva. Although, the same volume of glycerol was applied, the sclera cleared much faster (5 to 10 s) with the topical application of glycerol compared to the injection method (3 min). In addition, the direct topical application of glycerol spreads over a larger area in the sclera than the latter method. A diffuse optical spectroscopy system provided spectral analysis of the remitted light every two minutes during clearing and rehydration. Comparison of measurements to those obtained from phantoms with various absorption and scattering properties provided estimates of the absorption coefficient and reduced scattering coefficient of rabbit eye tissue.
L. Lim, Nichols, B., Rajaram, N., and Tunnell, J. W., “Probe pressure effects on human skin diffuse reflectance and fluorescence spectroscopy measurements.,” J Biomed Opt, vol. 16, no. 1, pp. 011012, 2011.Abstract
Diffuse reflectance and fluorescence spectroscopy are popular research techniques for noninvasive disease diagnostics. Most systems include an optical fiber probe that transmits and collects optical spectra in contact with the suspected lesion. The purpose of this study is to investigate probe pressure effects on human skin spectroscopic measurements. We conduct an in-vivo experiment on human skin tissue to study the short-term (<2 s) and long-term (>30 s) effects of probe pressure on diffuse reflectance and fluorescence measurements. Short-term light probe pressure (P0<9 mN∕mm2) effects are within 0 ± 10% on all physiological properties extracted from diffuse reflectance and fluorescence measurements, and less than 0±5% for diagnostically significant physiological properties. Absorption decreases with site-specific variations due to blood being compressed out of the sampled volume. Reduced scattering coefficient variation is site specific. Intrinsic fluorescence shows a large standard error, although no specific pressure-related trend is observed. Differences in tissue structure and morphology contribute to site-specific probe pressure effects. Therefore, the effects of pressure can be minimized when the pressure is small and applied for a short amount of time; however, long-term and large pressures induce significant distortions in measured spectra.