S. M. S. Kazmi, Parthasarthy, A. B., Song, N. E., Jones, T. A., and Dunn, A. K., “Chronic imaging of cortical blood flow using Multi-Exposure Speckle Imaging.,” Journal of cerebral blood flow and metabolism, vol. (accepted), 2013. Publisher's VersionAbstract
Chronic imaging of cerebral blood flow (CBF) is an important tool for investigating vascular remodeling after injury such as stroke. Although techniques such as Laser Speckle Contrast Imaging (LSCI) have emerged as valuable tools for imaging CBF in acute experiments, their utility for chronic measurements or cross-animal comparisons has been limited. Recently, an extension to LSCI called Multi-Exposure Speckle Imaging (MESI) was introduced that increases the quantitative accuracy of CBF images. In this paper, we show that estimates of chronic blood flow are better with MESI than with traditional LSCI. We evaluate the accuracy of the MESI flow estimates using red blood cell (RBC) photographic tracking as an absolute flow calibration in mice over several days. The flow measures computed using the MESI and LSCI techniques were found to be on average 10% and 24% deviant (n=9 mice), respectively, compared with RBC velocity changes. We also map CBF dynamics after photo-thrombosis of selected cortical microvasculature. Correlations of flow dynamics with RBC tracking were closer with MESI (r=0.88) than with LSCI (r=0.65) up to 2 weeks from baseline. With the increased quantitative accuracy, MESI can provide a platform for studying the efficacy of stroke therapies aimed at flow restoration.Journal of Cerebral Blood Flow & Metabolism advance online publication, 10 April 2013; doi:10.1038/jcbfm.2013.57.
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 in surgery and medicine, vol. 44, pp. 310–7, 2012. Publisher's VersionAbstract
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.
A. K. Dunn, “

Laser speckle contrast imaging of cerebral blood flow.

,” Annals of biomedical engineering, vol. 40, pp. 367–77, 2012. Publisher's VersionAbstract
Laser speckle contrast imaging (LSCI) has emerged over the past decade as a powerful, yet simple, method for imaging of blood flow dynamics in real time. The rapid adoption of LSCI for physiological studies is due to the relative ease and low cost of building an instrument as well as the ability to quantify blood flow changes with excellent spatial and temporal resolution. Although measurements are limited to superficial tissues with no depth resolution, LSCI has been instrumental in pre-clinical studies of neurological disorders as well as clinical applications including dermatological, neurosurgical and endoscopic studies. Recently a number of technical advances have been developed to improve the quantitative accuracy and temporal resolution of speckle imaging. This article reviews some of these recent advances and describes several applications of speckle imaging.
T. Wang, Mancuso, J. J., Kazmi, S. S. M., Dwelle, J., Sapozhnikova, V., Willsey, B., Ma, L. L., Qiu, J., Li, X., Dunn, A. K., Johnston, K. P., Feldman, M. D., and Milner, T. E., “

Combined two-photon luminescence microscopy and OCT for macrophage detection in the hypercholesterolemic rabbit aorta using plasmonic gold nanorose.

,” Lasers in surgery and medicine, vol. 44, pp. 49–59, 2012. Publisher's VersionAbstract
The macrophage is an important early cellular marker related to risk of future rupture of atherosclerotic plaques. Two-channel two-photon luminescence (TPL) microscopy combined with optical coherence tomography (OCT) was used to detect, and further characterize the distribution of aorta-based macrophages using plasmonic gold nanorose as an imaging contrast agent.
E. L. Towle, Richards, L. M., {Shams Kazmi}, S., Fox, D. J., and Dunn, A. K., “

Comparison of Indocyanine Green Angiography and Laser Speckle Contrast Imaging for the Assessment of Vasculature Perfusion.

,” Neurosurgery, 2012. Publisher's VersionAbstract
BACKGROUND:: Assessment of the vasculature is critical for overall success in cranial vascular neurological surgery procedures. While several methods of monitoring cortical perfusion intraoperatively are available, not all are appropriate or convenient in a surgical environment. Recently, two optical methods of care have emerged that are able to obtain high spatial resolution images with easily implemented instrumentation: indocyanine green (ICG) angiography and laser speckle contrast imaging (LSCI). OBJECTIVE:: To evaluate the usefulness of ICG and LSCI in measuring vessel perfusion. METHODS:: An experimental setup was developed that simultaneously collects measurements of ICG fluorescence and LSCI in a rodent model. A 785nm laser diode was used for both excitation of the ICG dye and the LSCI illumination. A photothrombotic clot model was used to occlude specific vessels within the field of view to enable comparison of the two methods for monitoring vessel perfusion. RESULTS:: The induced blood flow change demonstrated that ICG is an excellent method for visualizing the volume and type of vessel at a single point in time; however, it is not always an accurate representation of blood flow. In contrast, LSCI provides a continuous and accurate measurement of blood flow changes without the need of an external contrast agent. CONCLUSION:: These two methods should be used together in order to obtain a complete understanding of tissue perfusion.
E. L. Towle, Garcia, P. V., a Smith, P., Thomas, R. J., Dunn, A. K., Welch, A. J., and Foutch, B. K., “

Visual disruption using the thermal lensing effect in the human eye: pilot study.

,” Journal of biomedical optics, vol. 17, pp. 105007, 2012. Publisher's VersionAbstract
At select wavelengths, near infrared (IR) light is absorbed in the preretinal media of the eye. This produces small transient increases in temperature that temporarily alter the local index of refraction. If the IR exposure is sufficiently high, a momentary reduction in the focusing power of the eye can be induced through an effect known as thermal lensing. Fundamental optical interaction and safety aspects of this phenomenon have been demonstrated previously in animal and artificial eye models. However, whether the effect will induce an observable visual change in human subjects has not been explored. Here, results of a pilot study are shown where eight human subjects were exposed to an IR laser at levels that were below the safe exposure limit. The exposures did induce a transient visual distortion if sufficiently high levels were used. While the description of the visual change varied between subjects, this experiment was able to determine a general guideline for power needed to induce significant effects in human subjects.
A. K. Dunn, Leitgeb, R., Wang, R. K., and Zhang, H. F., “Introduction: feature issue on In Vivo Microcirculation Imaging.,” Biomedical Optics Express, vol. 2, pp. 1861–3, 2011. Publisher's VersionAbstract
The editors introduce the Biomedical Optics Express feature issue, "In Vivo Microcirculation Imaging," which includes 14 contributions from the biomedical optics community, covering such imaging techniques as optical coherence tomography, photoacoustic microscopy, laser Doppler /speckle imaging, and near infrared spectroscopy and fluorescence imaging.
R. V. Kuranov, Qiu, J., McElroy, A. B., Estrada, A., Salvaggio, A., Kiel, J., Dunn, A. K., Duong, T. Q., and Milner, T. E., “Depth-resolved blood oxygen saturation measurement by dual-wavelength photothermal (DWP) optical coherence tomography.,” Biomedical Optics Express, vol. 2, pp. 491–504, 2011. Publisher's VersionAbstract
Non-invasive depth-resolved measurement of hemoglobin oxygen saturation (SaO(2)) levels in discrete blood vessels may have implications for diagnosis and treatment of various pathologies. We introduce a novel Dual-Wavelength Photothermal (DWP) Optical Coherence Tomography (OCT) for non-invasive depth-resolved measurement of SaO(2) levels in a blood vessel phantom. DWP OCT SaO(2) is linearly correlated with blood-gas SaO(2) measurements. We demonstrate 6.3% precision in SaO(2) levels measured a phantom blood vessel using DWP-OCT with 800 and 765 nm excitation wavelengths. Sources of uncertainty in SaO(2) levels measured with DWP-OCT are identified and characterized.
K. Swanul, Dunn, A. K., Duong, T. Q., and Ress, D., “Measurements and modeling of transient blood flow perturbations induced by brief somatosensory stimulation.,” The open neuroimaging journal, vol. 5, pp. 96–104, 2011. Publisher's VersionAbstract
Proper interpretation of BOLD fMRI and other common functional imaging methods requires an understanding of neurovascular coupling. We used laser speckle-contrast optical imaging to measure blood-flow responses in rat somatosensory cortex elicited by brief (2 s) forepaw stimulation. Results show a large increase in local blood flow speed followed by an undershoot and possible late-time oscillations. The blood flow measurements were modeled using the impulse response of a simple linear network, a four-element windkessel. This model yielded excellent fits to the detailed time courses of activated regions. The four-element windkessel model thus provides a simple explanation and interpretation of the transient blood-flow response, both its initial peak and its late-time behavior.
M. A. Davis, {Shams Kazmi}, S., Ponticorvo, A., and Dunn, A. K., “Depth dependence of vascular fluorescence imaging.,” Biomedical optics express, vol. 2, pp. 3349–62, 2011. Publisher's VersionAbstract
In vivo surface imaging of fluorescently labeled vasculature has become a widely used tool for functional brain imaging studies. Techniques such as phosphorescence quenching for oxygen tension measurements and indocyanine green fluorescence for vessel perfusion monitoring rely on surface measurements of vascular fluorescence. However, the depth dependence of the measured fluorescence signals has not been modeled in great detail. In this paper, we investigate the depth dependence of the measured signals using a three-dimensional Monte Carlo model combined with high resolution vascular anatomy. We found that a bulk-vascularization assumption to modeling the depth dependence of the signal does not provide an accurate picture of penetration depth of the collected fluorescence signal in most cases. Instead the physical distribution of microvasculature, the degree of absorption difference between extravascular and intravascular space, and the overall difference in absorption at the excitation and emission wavelengths must be taken into account to determine the depth penetration of the fluorescence signal. Additionally, we found that using targeted illumination can provide for superior surface vessel sensitivity over wide-field illumination, with small area detection offering an even greater amount of sensitivity to surface vasculature. Depth sensitivity can be enhanced by either increasing the detector area or increasing the illumination area. Finally, we see that excitation wavelength and vessel size can affect intra-vessel sampling distribution, as well as the amount of signal that originates from inside the vessel under targeted illumination conditions.
R. V. Kuranov, Kazmi, S., McElroy, A. B., Kiel, J. W., Dunn, A. K., Milner, T. E., and Duong, T. Q., “In vivo depth-resolved oxygen saturation by Dual-Wavelength Photothermal (DWP) OCT.,” Optics express, vol. 19, pp. 23831–44, 2011. Publisher's VersionAbstract
Microvasculature hemoglobin oxygen saturation (SaO2) is important in the progression of various pathologies. Non-invasive depth-resolved measurement of SaO2 levels in tissue microvasculature has the potential to provide early biomarkers and a better understanding of the pathophysiological processes allowing improved diagnostics and prediction of disease progression. We report proof-of-concept in vivo depth-resolved measurement of SaO(2) levels in selected 30 µm diameter arterioles in the murine brain using Dual-Wavelength Photothermal (DWP) Optical Coherence Tomography (OCT) with 800 nm and 770 nm photothermal excitation wavelengths. Depth location of back-reflected light from a target arteriole was confirmed using Doppler and speckle contrast OCT images. SaO(2) measured in a murine arteriole with DWP-OCT is linearly correlated (R(2)=0.98) with systemic SaO(2) values recorded by a pulse-oximeter. DWP-OCT are steadily lower (10.1%) than systemic SaO(2) values except during pure oxygen breathing. DWP-OCT is insensitive to OCT intensity variations and is a candidate approach for in vivo depth-resolved quantitative imaging of microvascular SaO(2) levels.
H. Karatas, Erdener, S. E., Gursoy-Ozdemir, Y., Gurer, G., Soylemezoglu, F., Dunn, A. K., and Dalkara, T., “Thrombotic distal middle cerebral artery occlusion produced by topical FeCl(3) application: a novel model suitable for intravital microscopy and thrombolysis studies.,” Journal of cerebral blood flow and metabolism, vol. 31, pp. 1452–60, 2011. Publisher's VersionAbstract
Intravital or multiphoton microscopy and laser-speckle imaging have become popular because they allow live monitoring of several processes during cerebral ischemia. Available rodent models have limitations for these experiments; e.g., filament occlusion of the proximal middle cerebral artery (MCA) is difficult to perform under a microscope, whereas distal occlusion methods may damage the MCA and the peri-arterial cortex. We found that placement of a 10% FeCl(3)-soaked filter paper strip (0.3 × 1 mm(2)) on the duramater over the trunk of the distal MCA through a cranial window for 3 minutes induced intraarterial thrombus without damaging the peri-arterial cortex in the mouse. This caused a rapid regional cerebral blood flow decrease within 10 minutes and total occlusion of the MCA segment under the filter paper in 17±2 minutes, which resulted in a typical cortical infarct of 27±4 mm(3) at 24 hours and moderate sensorimotor deficits. There was no significant hemispheric swelling or hemorrhage or mortality at 24 hours. Reperfusion was obtained in half of the mice with tissue plasminogen activator, which allowed live monitoring of clot lysis along with restoration of tissue perfusion and MCA flow. In conclusion, this relatively simple and noninvasive stroke model is easy to perform under a microscope, making it suitable for live imaging and thrombolysis studies.
A. D. Estrada and Dunn, A. K., “Improved sensitivity for two-photon frequency-domain lifetime measurement.,” Optics Express, vol. 18, pp. 13631–9, 2010. Publisher's VersionAbstract
We demonstrate a method to improve the measurement sensitivity of two-photon frequency-domain lifetime measurements in poor signal to background conditions. This technique uses sinusoidal modulation of the two-photon excitation source and detection of the second harmonic of the modulation frequency that appears in the emission. Additionally, we present the mathematical model which describes how the observed phase shift and amplitude demodulation factor of two-photon phosphorescence emission are related to the phosphorescence lifetime and modulation frequency. We demonstrate the validity of the model by showing the existence of new frequency terms in the phosphorescence emission generated from the quadratic nature of two-photon absorption and by showing that the phase shift and demodulation match theory for all frequency components.
H. Nakamura, Strong, A. J., Dohmen, C., Sakowitz, O. W., Vollmar, S., Sué, M., Kracht, L., Hashemi, P., Bhatia, R., Yoshimine, T., Dreier, J. P., Dunn, A. K., and Graf, R., “Spreading depolarizations cycle around and enlarge focal ischaemic brain lesions.,” Brain, vol. 133, pp. 1994–2006, 2010. Publisher's VersionAbstract
How does infarction in victims of stroke and other types of acute brain injury expand to its definitive size in subsequent days? Spontaneous depolarizations that repeatedly spread across the cerebral cortex, sometimes at remarkably regular intervals, occur in patients with all types of injury. Here, we show experimentally with in vivo real-time imaging that similar, spontaneous depolarizations cycle repeatedly around ischaemic lesions in the cerebral cortex, and enlarge the lesion in step with each cycle. This behaviour results in regular periodicity of depolarization when monitored at a single point in the lesion periphery. We present evidence from clinical monitoring to suggest that depolarizations may cycle in the ischaemic human brain, perhaps explaining progressive growth of infarction. Despite their apparent detrimental role in infarct growth, we argue that cycling of depolarizations around lesions might also initiate upregulation of the neurobiological responses involved in repair and remodelling.
A. B. Parthasarathy, Kazmi, S. S. M., and Dunn, A. K., “Quantitative imaging of ischemic stroke through thinned skull in mice with Multi Exposure Speckle Imaging.,” Biomedical Optics Express, vol. 1, pp. 246–259, 2010. Publisher's VersionAbstract
Laser Speckle Contrast Imaging (LSCI) has become a widely used technique to image cerebral blood flow in vivo. However, the quantitative accuracy of blood flow changes measured through the thin skull has not been investigated thoroughly. We recently developed a new Multi Exposure Speckle Imaging (MESI) technique to image blood flow while accounting for the effect of scattering from static tissue elements. In this paper we present the first in vivo demonstration of the MESI technique. The MESI technique was used to image the blood flow changes in a mouse cortex following photothrombotic occlusion of the middle cerebral artery. The Multi Exposure Speckle Imaging technique was found to accurately estimate flow changes due to ischemia in mice brains in vivo. These estimates of these flow changes were found to be unaffected by scattering from thinned skull.
A. Ponticorvo and Dunn, A. K., “How to build a Laser Speckle Contrast Imaging (LSCI) system to monitor blood flow.,” Journal of visualized experiments : JoVE, 2010. Publisher's VersionAbstract
Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.
A. B. Parthasarathy, Weber, E. L., Richards, L. M., Fox, D. J., and Dunn, A. K., “Laser speckle contrast imaging of cerebral blood flow in humans during neurosurgery: a pilot clinical study,” Journal of Biomedical Optics, vol. 15, pp. 066030, 2010. Publisher's VersionAbstract
Monitoring cerebral blood flow (CBF) during neurosurgery can provide important physiological information for a variety of surgical procedures. CBF measurements are important for assessing whether blood flow has returned to presurgical baseline levels and for assessing postsurgical tissue viability. Existing techniques for intraoperative monitoring of CBF based on magnetic resonance imaging are expensive and often impractical, while techniques such as indocyanine green angiography cannot produce quantitative measures of blood flow. Laser speckle contrast imaging (LSCI) is an optical technique that has been widely used to quantitatively image relative CBF in animal models in vivo. In a pilot clinical study, we adapted an existing neurosurgical operating microscope to obtain LSCI images in humans in real time during neurosurgery under baseline conditions and after bipolar cautery. Simultaneously recorded ECG waveforms from the patient were used to develop a filter that helped reduce measurement variabilities due to motion artifacts. Results from this study demonstrate the feasibility of using LSCI to obtain blood flow images during neurosurgeries and its capability to produce full field CBF image maps with excellent spatial resolution in real-time with minimal disruption to the surgical procedure.
A. Ponticorvo and Dunn, A. K., “Simultaneous imaging of oxygen tension and blood flow in animals using a digital micromirror device.,” Optics Express, vol. 18, pp. 8160–70, 2010. Publisher's VersionAbstract
In this study we present a novel imaging method that combines high resolution cerebral blood flow imaging with a highly flexible map of absolute pO(2). In vivo measurements of pO(2) in animals using phosphorescence quenching is a well established method, and is preferable over electrical probes which are inherently invasive and are limited to single point measurements. However, spatially resolved pO(2) measurements using phosphorescence lifetime quenching typically require expensive cameras to obtain images of pO(2) and often suffer from poor signal to noise. Our approach enables us to retain the high temporal resolution and sensitivity of single point detection of phosphorescence by using a digital micromirror device (DMD) to selectively illuminate arbitrarily shaped regions of tissue. In addition, by simultaneously using Laser Speckle Contrast Imaging (LSCI) to measure relative blood flow, we can better examine the relationship between blood flow and absolute pO(2). We successfully used this instrument to study changes that occur during ischemic conditions in the brain with enough spatial resolution to clearly distinguish different regions. This novel instrument will provide researchers with an inexpensive and improved technique to examine multiple hemodynamic parameters simultaneously in the brain as well as other tissues.
M. S. Starosta and Dunn, A. K., “Far-field superposition method for three-dimensional computation of light scattering from multiple cells.,” Journal of Biomedical Optics, vol. 15, pp. 055006, 2010. Publisher's VersionAbstract
A linear coherent superposition method for estimating the plane wave far-field scattering pattern from multiple biological cells computed by the finite-difference time-domain (FDTD) method is presented. The method enables the FDTD simulation results of scattering from a small number of complex scatterers, such as biological cells, to be used to estimate the far-field pattern from a large group of those same scatterers. The superposition method can be used to reduce the computational cost of FDTD simulations by enabling a single large scattering problem to be broken into smaller problems with more practical computational requirements. It is found that the method works best in cases where there is little multiple scattering interaction between adjacent cells, so the far-field pattern of multicell geometry can simply be calculated as a phase-adjusted linear superposition of the scattering from individual cells. A strategy is also presented for choosing the minimum number of cells in cases with significant multiple scattering interactions between cells.
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 in surgery and medicine, vol. 42, pp. 630–639, 2010. Publisher's VersionAbstract
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 $μ$m 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 $μ$m 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.