Two-photon imaging of blood flow in the rat cortex.

Cerebral blood flow plays a central role in maintaining homeostasis in the brain, and its dysfunction leads to pathological conditions such as stroke. Moreover, understanding the dynamics of blood flow is central to the interpretation of data from imaging modalities--such as intrinsic optical signaling and functional magnetic resonance imaging--that rely on changes in cerebral blood flow and oxygen level to infer changes in the underlying neural activity. Recent advances in imaging techniques have allowed detailed studies of blood flow in vivo at high spatial and temporal resolutions. We discuss techniques to accurately measure cerebral blood flow at the level of individual blood vessels using two-photon laser-scanning microscopy. By directing the scanning laser along a user-defined path, it is possible to measure red blood cell (RBC) velocity and vessel diameter across multiple vessels simultaneously. The combination of these measurements permits accurate assessment of total flux with sufficient time resolution to measure fast modulations in flux, such as those caused by heartbeat, as well as slower signals caused by vasomotion and hemodynamic responses to stimulus. Here, we discuss general techniques for animal preparation and measurement of blood flow with two-photon microscopy. We incorporate extensions to existing methods to accurately acquire flux data simultaneously across multiple vessels in a single trial. Central to these measurements is the ability to generate scan paths that smoothly connect user-defined lines of interest while maintaining high accuracy of the scan path.

Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.

The cerebral vascular system services the constant demand for energy during neuronal activity in the brain. Attempts to delineate the logic of neurovascular coupling have been greatly aided by the advent of two-photon laser scanning microscopy to image both blood flow and the activity of individual cells below the surface of the brain. Here we provide a technical guide to imaging cerebral blood flow in rodents. We describe in detail the surgical procedures required to generate cranial windows for optical access to the cortex of both rats and mice and the use of two-photon microscopy to accurately measure blood flow in individual cortical vessels concurrent with local cellular activity. We further provide examples on how these techniques can be applied to the study of local blood flow regulation and vascular pathologies such as small-scale stroke.

A guide to delineate the logic of neurovascular signaling in the brain.

The neurovascular system may be viewed as a distributed nervous system within the brain. It transforms local neuronal activity into a change in the tone of smooth muscle that lines the walls of arterioles and microvessels. We review the current state of neurovascular coupling, with an emphasis on signaling molecules that convey information from neurons to neighboring vessels. At the level of neocortex, this coupling is mediated by: (i) a likely direct interaction with inhibitory neurons, (ii) indirect interaction, via astrocytes, with excitatory neurons, and (iii) fiber tracts from subcortical layers. Substantial evidence shows that control involves competition between signals that promote vasoconstriction versus vasodilation. Consistent with this picture is evidence that, under certain circumstances, increased neuronal activity can lead to vasoconstriction rather than vasodilation. This confounds naïve interpretations of functional brain images. We discuss experimental approaches to detect signaling molecules in vivo with the goal of formulating an empirical basis for the observed logic of neurovascular control.

Photon counting, censor corrections, and lifetime imaging for improved detection in two-photon microscopy.

We present a high-speed photon counter for use with two-photon microscopy. Counting pulses of photocurrent, as opposed to analog integration, maximizes the signal-to-noise ratio so long as the uncertainty in the count does not exceed the gain-noise of the photodetector. Our system extends this improvement through an estimate of the count that corrects for the censored period after detection of an emission event. The same system can be rapidly reconfigured in software for fluorescence lifetime imaging, which we illustrate by distinguishing between two spectrally similar fluorophores in an in vivo model of microstroke.

Chronic optical access through a polished and reinforced thinned skull.

We present a method to form an optical window in the mouse skull that spans millimeters and is stable for months without causing brain inflammation. This enabled us to repeatedly image blood flow in cortical capillaries of awake mice and determine long-range correlations in speed. We also repeatedly imaged dendritic spines, microglia and angioarchitecture, as well as used illumination to drive motor output via optogenetics and induce microstrokes via photosensitizers.

Automatic identification of fluorescently labeled brain cells for rapid functional imaging.

The on-line identification of labeled cells and vessels is a rate-limiting step in scanning microscopy. We use supervised learning to formulate an algorithm that rapidly and automatically tags fluorescently labeled somata in full-field images of cortex and constructs an optimized scan path through these cells. A single classifier works across multiple subjects, regions of the cortex of similar depth, and different magnification and contrast levels without the need to retrain the algorithm. Retraining only has to be performed when the morphological properties of the cells change significantly. In conjunction with two-photon laser scanning microscopy and bulk-labeling of cells in layers 2/3 of rat parietal cortex with a calcium indicator, we can automatically identify ∼ 50 cells within 1 min and sample them at ∼ 100 Hz with a signal-to-noise ratio of ∼ 10.