The penetration depth of the infrared laser depends on the optical properties of (a) the laser light and (b) the sampled tissue and is reported in the range of 500 μm to 1 mm (6, 9). In general, tissue perfusion and flow velocity in large vessels can be measured in exposed tissue such as the human brain, where the vasculature courses close to the surface (7). In the mouse, after retraction of the scalp cortical perfusion can be easily assessed through the intact scull without the need for drilling a cranial window (1).

The spatial and temporal resolution is inversely related and its selection is a compromise of acquiring sharp and detailed images with a sufficiently high sampling rate for dynamic real-time live imaging. For continuous high-resolution transcranial assesment of the cerebrovascular reactivity by acetazolamide challenge in mice (see chapter 2.1.4 and Fig. 2), we recommend a sampling rate between 1 and 0.25 Hz, depending on the duration of the entire measurement.

Laser speckle imaging delivers an immediate, noninvasive functional readout of relative cortical blood flow with direct assessment of tissue perfusion. In order to plot flux values over time in a graph or chart, regions of interest (ROI) ROIs with variable size and location can be positioned over the speckle images and the CBF-flux within that ROI is recorded for each image at the same sampling rate as the live image measurement. The flux value within this user-defined ROI is a calculated mean of the individual flux values within the region. The values are plotted and the resultant trace can be analyzed to compare flow changes before, during, and after an intervention. Figure 2a, b gives an example of in vivo LASCA images during cerebraovascular reactivity testing (Fig. 2a) and plotted CBF-flux (Fig. 2b) 6 h after experimental SAH in a C57/BL6 mouse.