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Multiphoton Microscopy

The human brain receives twenty percent of cardiac output. Resultantly, the blood supply meets approximately 2.5 times the average metabolic demand of the brain. The main payload of this blood supply is molecular oxygen bound to erythrocytes and dissociated into plasma, where both short- and long-term deprivation can have significant physiological impact. During a stroke, the supply of blood carrying the oxygen is severely reduced or completely diminished, resulting in decreased neuronal function and cell death from ischemia despite the seemingly global oversupply versus metabolic demand. Therefore, understanding the bottlenecks in supply is vital for characterizing the cerebrovascular plumbing, which can benefit from both quantitative and high resolution mapping.

Unraveling the cerebrovascular architecture is an active area of physiological research, currently only understood from the perspective of large regional supply routes dominated by specific arterial branches of the carotid arteries. In brief, supply routes stemming from the meninges feed the cerebral cortex, where branches of the anterior, medial, and posterior cerebral arteries supply large segments of the functional gray matter. More locally, arterioles are critical to supplying the cortical microvascular network, which begin as pial arterioles on the surface and then dive into the cortical layers. These vessels are responsible for oxygenating the gray matter and predominantly return to draining venules on the cortical surface.

We utilize multiphoton fluorescence and phosphorescence lifetime microscopy to render the microvascular architecture and dissolved oxygen concentrations, respectively, in vessels supplying the cortical layers of the mouse brain in vivo. The nonlinear imaging technique provides exceptionally localized excitation within the tissue volume, enabling three-dimensional imaging by scanning and stepping the illumination in space. By using fluorescent contrast agents and proteins both the vascular and tissue environments can be labeled precisely in vivo. Beyond providing contrast, specialized phosphorescent probes can be utilized to optically sense the local dissolved oxygen tension (pO2) within the nonlinear excitation volume. Given the precise excitation technique, sensitive detection elements and electronics are utilized to register the emission signals with high SNR and temporal resolution.

Relevant Publications

In vivo multiphoton imaging of a diverse array of fluorophores to investigate deep neurovascular structure

D. R. Miller, A. M. Hassan, J. W. Jarrett, F. A. Medina, E. P. Perillo, K. Hagan, S. M. S. Kazmi, T. A. Clark, C. T. Sullender, T. A. Jones, B. V. Zemelman, and A. K. Dunn, Biomedical Optics Express (2017)

Deep in vivo two­-photon microscopy with a low cost custom built mode-­locked 1060 nm fiber laser

E. P. Perillo, J. E. McCracken, D. C. Fernée, J. R. Goldak, F. A. Medina, D. R. Miller, H. C. Yeh, and A. K. Dunn, Biomedical Optics Express (2016)

Chronic monitoring of vascular progression after ischemic stroke using multiexposure speckle imaging and two-photon fluorescence microscopy.

C. J. Schrandt, S. M. S. Kazmi, T. A. Jones, and A. K. Dunn, Journal of Cerebral Blood Flow & Metabolism (2015)

Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion

S. M. S. Kazmi, A. J. Salvaggio, A. D. Estrada, M. A. Hemati, N. K. Shaydyuk, E. Roussakis, T. A. Jones, S. A. Vinogradov, and A. K. Dunn, Biomedical Optics Express (2013)

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

T. Wang, J. J. Mancuso, S. M. S. Kazmi, J. Dwelle, V. Sapozhnikova, B. Willsey, L. L. Ma, J. Qiu, X. Li, A. K. Dunn, K. P. Johnston, M. D. Feldman, and T. E. Milner, Lasers in Surgery and Medicine (2012)

Intra-organ biodistribution of gold nanoparticles using intrinsic two-photon-induced photoluminescence

J. Park, A. Estrada, J. A. Schwartz, P. Diagaradjane, S. Krishnan, A. K. Dunn, and J. W. Tunnell, Lasers in Surgery and Medicine (2010)

Improved sensitivity for two-photon frequency-domain lifetime measurement

A. D. Estrada and A. K. Dunn, Optics Express (2010)

Microvascular oxygen quantification using two-photon microscopy

A. D. Estrada, A. Ponticorvo, T. N. Ford, and A. K. Dunn, Optics Letters (2008)

Two-photon-induced photoluminescence imaging of tumors using near-infrared excited gold nanoshells

J. Park, A. Estrada, K. Sharp, K. Sang, J. A. Schwartz, D. K. Smith, C. Coleman, J. D. Payne, B. A. Korgel, A. K. Dunn, and J. W. Tunnell, Optics Express (2008)

Depth-resolved optical imaging and microscopy of vascular compartment dynamics during somatosensory stimulation

E. Hillman, A. Devor, M. B. Bouchard, A. K. Dunn, G. W. Krauss, J. Skoch, B. J. Bacskai, A. M. Dale, and D. A. Boas, NeuroImage (2007)

Influence of optical properties on two-photon fluorescence imaging in turbid samples

A. K. Dunn, V. P. Wallace, M. Coleno, M. W. Berns, and B. J. Tromberg, Applied Optics (2000)
Two-Photon Image of Cortical Vasculature

This maximum intensity projection of mouse cortical vasculature was stitched together using four separate two-photon z-stacks for a total imaging volume of 1000µm x 1000µm x 500µm. (Davis, et al., 2014)

Two-Photon Microscope Schematic

Custom-built two-photon microscope and laser speckle contrast imaging system with time-correlated single photon counting capabilities. (Kazmi, et al., 2013)

3D Mapping of Oxygen Tension

Two-photon phosphorescence lifetime imaging is used to characterize the oxygen tension (pO2) in individual cortical vessels. As arterioles descend deeper into the brain, the pO2 rapidly decreases. (Kazmi, et al., 2013)

Depth-Resolved Vascular Imaging

Multi-photon excitation allows for deep imaging of cortical vasculature far beyond the limits of other optical techniques such as confocal microscopy. This z-stack of fluorescently-labeled blood vessels spans 200µm x 200µm x 1200µm.