Abstract
Neurovascular coupling, cerebrovascular remodeling and hemodynamic changes are critical to brain function, and dysregulated in neuropathologies such as brain tumors. Interrogating these phenomena in freely behaving animals requires a portable microscope with multiple optical contrast mechanisms. Therefore, we developed a miniaturized microscope with: a fluorescence (FL) channel for imaging neural activity (e.g., GCaMP) or fluorescent cancer cells (e.g., 9L-GFP); an intrinsic optical signal (IOS) channel for imaging hemoglobin absorption (i.e., cerebral blood volume); and a laser speckle contrast (LSC) channel for imaging perfusion (i.e., cerebral blood flow). Following extensive validation, we demonstrate the microscope’s capabilities via experiments in unanesthetized murine brains that include: (i) multi-contrast imaging of neurovascular changes following auditory stimulation; (ii) wide-area tonotopic mapping; (iii) EEG-synchronized imaging during anesthesia recovery; and (iv) microvascular connectivity mapping over the life-cycle of a brain tumor. This affordable, flexible, plug-and-play microscope heralds a new era in functional imaging of freely behaving animals.
Highlights
Idealized cerebral blood flow (CBF)5 μm pixel time course responseIdealized response
Speckle contrast is computed from images acquired under red laser diode illumination, enabling us to map in vivo cerebral blood flow (CBF)
There is an exigent need for imaging tools capable of simultaneously interrogating multiple physiological variables ranging from neural activity to hemodynamics, while being synchronized with external electrophysiological monitoring equipment, and without the confounding effect of anesthetics
Summary
Miniature plug-and-play design with three optical contrast mechanisms. In contrast to previously reported high-magnification/small field of view (FoV) miniaturized microscope designs[6,7,22], ours trades magnification in favor of a larger (3 × 3 mm2) FoV to ensure greater cortical coverage. The stimulus-evoked blood-oxygen-leveldependent (BOLD) fMRI signal is treated as a surrogate of neuronal activation and used as an input to a pixel-wise General Linear Model (GLM) to identify activated pixels (see Methods) This process involves modeling the BOLD response observed in each pixel as a combination of an idealized response and background signal (Fig. 4a). We used the Analysis of Functional Neuro Images (AFNI)[38] fMRI processing software to generate GLM-based wide-area activation maps of auditory cortical tonotopy by exploiting the multi-contrast capabilities of the microscope. For our multi-contrast GLM approach, the map of the neuronal response (i.e., GCaMP fluorescence) to the 4 kHz stimulus served as the gold standard for identifying activated brain areas.
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