Neuroimaging

Using light, we are able to image the living brain in a number of different ways to be able to visualize both the neuronal and hemodynamic behavior of the brain at both ensemble and cellular levels. Optical neuroimaging is a major focus of our lab, and in particular, we are seeking to use our imaging systems to better understand neurovascular coupling.

What is Neurovascular Coupling?

A local increase in cortical blood flow accompanies almost all neuronal responses to stimulus in the brain. This hemodynamic response is the basis of functional magnetic resonance imaging (fMRI). However, surprisingly little is understood about the interrelation between blood flow and the neuronal activity that underlies it. It would be of significant value to be able to interpret clinical fMRI data in terms of neuronal function. Yet more importantly, normal functioning of the brain seems to depend critically on the integrity of neurovascular coupling, so understanding the mechanistic basis of this coupling could yield therapeutic targets for a range of pathologies including Alzheimer's and age related neurodegeneration.

Figure 1. Left: Elements of the neurovascular unit. Astrocytes, interneurons, pericytes and the blood vessels themselves are all suspected to play a role in orchestrating the hemodynamic response to stimulus. *Adapted from: Gordon, Mulligan & MacVicar, Glia ,2007. Right: in-vivo two-photon image of the interactions between blood vessels and astrocytes.

Studying neurovascular coupling is complex owing to the need for both neuronal interconnectivity and vascular networks to be intact. In-vivo imaging allows the complete neurovascular system to be observed and perturbed and directly linked to clinical measures such as fMRI. However in-vivo imaging presents significant challenges in terms of experimental paradigm and instrument design. Advanced imaging techniques are required to capture the behavior of the brain in real time, measuring both vascular and neuronal parameters in parallel, and probing beneath the surface of the cortex to resolve 3D and layer-specific interactions. To date we have applied three technologies to in-vivo imaging of neurovascular coupling; 1) In-vivo two-photon microscopy, 2) High-speed multispectral camera imaging and 3) Laminar optical tomography for depth-resolved imaging.

In-vivo two-photon microscopy of neurovascular coupling

Two-photon microscopy allows in-vivo imaging of fluorescent contrast to depths of >500 microns in the living brain. We are using two-photon imaging to investigate the cellular-level interactions between neurons and blood vessels during normal responses to somatosensory stimulus in rodents. Several different types of cells are implicated in neurovascular control including astrocytes and interneurons. We can specifically target these cells in-vivo using transgenic models expressing GFP or other fluorescent proteins in specific cell types, or cell-specific dyes such as sulforhodamine SR101 (specific to astrocytes). Vasculature can be highlighted using dextran-conjugated dyes injected intravascularly, which also allow changes in vessel diameter and blood flow to be evaluated. Calcium sensitive dyes can also be used in-vivo and imaged using two-photon microscopy, providing direct cellular-level read-outs of neuronal and astrocytic activation. We have designed our two-photon microscope to image many of these forms of contrast in parallel and at high speed to capture the evolution of neurovascular coupling at the level of single cells and vessels.

Multispectral camera imaging of neurovascular coupling

'Intrinsic signal imaging' has been used for over 20 years to examine cortical responses to stimulus. In its simplest form, such imaging requires only a light source at a specific wavelength, and a camera to detect changes in the amount of light remitted from the brain's surface. It is now widely understood that changes in reflectance of the brain are due to changes in the local concentration of absorbing oxy- and deoxy-hemoglobin. It is therefore possible to make 'intrinsic signal imaging' more quantitative if multiple wavelengths of illumination are used, and modeling of light propagation is employed to convert measurements of light intensity into changes in oxy- and deoxy-hemoglobin. A further extension of this kind of imaging is to add fluorescent voltage or calcium sensitive dyes to the brain to allow equivalent imaging of neuronal activity.

We have developed a high-speed camera-based imaging system to allow detailed characterization of the hemodynamic response of the brain to stimulus. Our system can acquire between 50 and 100 frames per second at two or more wavelengths, or acquire interlaced reflectance and fluorescence data. We combine use of this system with in-vivo two-photon microscopy to allow us to investigate and characterize the ensemble response of the cortex to stimulus, followed by detailed dynamic investigation of cellular behavior in targeted regions.

Figure 3. Left: camera-based imaging of the ensemble response to 3Hz forepaw stimulus in the exposed somatosensory cortex. Right: high-speed two-photon imaging of the same response in the region shown. See Hillman 2007 for more details.

3D cortical imaging using LOT

Laminar Optical Tomography is a technique that we developed to image depth-resolved functional activity in exposed cortex. LOT uses measurements of scattered light to add a depth-dimension to conventional multi-spectral optical brain imaging. LOT can currently image hemodynamic activity to > 2mm, with 100-200 micron resolution at over 80 frames per second. For more details on the LOT system see here.

To date, we have demonstrated that LOT can resolve the oxy– and deoxy-hemoglobin responses in individual vascular compartments in the somatosensory cortex during forepaw stimulation. This was achieved using a unique spatiotemporal linear fitting procedure based on the characteristic temporal behavior of the arteriolar, capillary and venous responses. We have now added fluorescence imaging capabilities to LOT to allow simultaneous depth-resolved imaging of voltage or calcium sensitive fluorescent dyes to enable 3D studies of neurovascular coupling in the living brain through the full thickness of the cortex.

Figure 4. Laminar Optical Tomography of the hemodynamic response to forepaw stimulus in the rodent somatosensory cortex (thinned skull). For more details see Hillman et al 2007.

Related Publications

Hillman E. M. C, Devor A, Bouchard M. B, Dunn A. K, Krauss GW, Skoch J, Bacskai J, Dale A. M, Boas D. A. “Depth-resolved Optical Imaging and Microscopy of Vascular Compartment Dynamics During Somatosensory Stimulation”, NeuroImage, 35(1): 89-104 (2007)

Bouchard M. B., Chen B. R, Burgess S. A, Hillman E. M. C, "Ultra-fast multispectral optical imaging of cortical oxygenation and blood flow dynamics", Optics Express, 17 (18), 15670-15678, (2009).

Hillman E. M. C, “Optical Brain Imaging In-vivo: Techniques and Applications from Animal to Man” [Invited Review] J Biomed Opt, 12(5), 051402 (2007). *Also selected to appear in the “Virtual Journal of Biological Physics Research”

Karagiannis A, Gallopin T, David C, Battaglia D, Geoffroy H, Rossier J, Hillman E.M.C, Staiger J, Cauli B. “Classification of NPY-expressing neocortical interneurons", J. Neurosci, 29(11):3642-3659 (2009).

Devor A, Hillman E. M. C, Tian P, Waeber C, Teng I. C, Ruvinskaya S, Shalinsky M, Zhu H, Haslinger R, Narayanan S, Ulbert I, Dunn A. K, Lo E, Rosen B. R, Dale A. M, Kleinfeld D, Boas D. A, “Stimulus-induced changes in blood flow and 2-deoxyglucose uptake dissociate in ipsilateral somatosensory cortex", J. Neurosci, 28: 14347-14357 (2008).

Devor A, Tian P, Nishimura N, Teng IC, Hillman E. M. C, Narayanan S. N, Ulbert I, Boas D. A, Kleinfeld D, Dale A. M. “Suppressed neuronal activity and concurrent arteriolar vasoconstriction may explain negative BOLD”, J Neurosci, 27(16): 4452-4459. (2007)

Hillman E. M. C,  Burgess S. A, Chen B. “Macroscopic molecular optical imaging of rodent brain”, Book Chapter in ‘Molecular Imaging of the Rodent Brain’ (Neuromethods Series), Editor. Bacskai B. J, Ed. In press 2007, Human Press (Springer).

Hillman E. M. C, Bouchard M, Devor A, De Crespigny A, Boas D. A. “Functional optical imaging of brain activation: a multi-scale, multi-modality approach” IEEE Proceedings of the Life Science Systems and Application Workshop, Bethesda MD, July 2006.

Radosevich A. J, Bouchard M. B, Burgess S. A, Chen B. R, Hillman E. M. C. “Hyper-spectral in-vivo two-photon microscopy of intrinsic fluorophores”, Optics Letters, 33 (18), 2164-2166, (2008)

Burgess S. A, Bouchard M. B, Yuan B, Hillman E. M. C. “Simultaneous Multi-Wavelength Laminar Optical Tomography"Optics Letters, 33 (22), 2710-2712 (2008).