Fast confocal fluorescence imaging in freely behaving mice.

Fluorescence imaging in the brain of freely behaving mice is challenging due to severe miniaturization constraints. In particular, the ability to image a large field of view at high temporal resolution and with efficient out-of-focus background rejection still raises technical difficulties. Here, we present a novel fiberscope system that provides fast (up to 200 Hz) background-free fluorescence imaging in freely behaving mice over a field of view of diameter 230 µm. The fiberscope is composed of a custom-made multipoint-scanning confocal microscope coupled to the animal with an image guide and a micro-objective. By simultaneously registering a multipoint-scanning confocal image and a conventional widefield image, we subtracted the residual out-of-focus background and provided a background-free confocal image. Illumination and detection pinholes were created using a digital micromirror device, providing high adaptability to the sample structure and imaging conditions. Using this novel imaging tool, we demonstrated fast fluorescence imaging of microvasculature up to 120 µm deep in the mouse cortex, with an out-of-focus background reduced by two orders of magnitude compared with widefield microscopy. Taking advantage of the high acquisition rate (200 Hz), we measured red blood cell velocity in the cortical microvasculature and showed an increase in awake, unrestrained mice compared with anaesthetized animals.

Mapping the dynamics of brain perfusion using functional ultrasound in a rat model of transient middle cerebral artery occlusion.

Following middle cerebral artery occlusion, tissue outcome ranges from normal to infarcted depending on depth and duration of hypoperfusion as well as occurrence and efficiency of reperfusion. However, the precise time course of these changes in relation to tissue and behavioral outcome remains unsettled. To address these issues, a three-dimensional wide field-of-view and real-time quantitative functional imaging technique able to map perfusion in the rodent brain would be desirable. Here, we applied functional ultrasound imaging, a novel approach to map relative cerebral blood volume without contrast agent, in a rat model of brief proximal transient middle cerebral artery occlusion to assess perfusion in penetrating arterioles and venules acutely and over six days thanks to a thinned-skull preparation. Functional ultrasound imaging efficiently mapped the acute changes in relative cerebral blood volume during occlusion and following reperfusion with high spatial resolution (100 µm), notably documenting marked focal decreases during occlusion, and was able to chart the fine dynamics of tissue reperfusion (rate: one frame/5 s) in the individual rat. No behavioral and only mild post-mortem immunofluorescence changes were observed. Our study suggests functional ultrasound is a particularly well-adapted imaging technique to study cerebral perfusion in acute experimental stroke longitudinally from the hyper-acute up to the chronic stage in the same subject.

Real-time imaging of brain activity in freely moving rats using functional ultrasound.

Innovative imaging methods help to investigate the complex relationship between brain activity and behavior in freely moving animals. Functional ultrasound (fUS) is an imaging modality suitable for recording cerebral blood volume (CBV) dynamics in the whole brain but has so far been used only in head-fixed and anesthetized rodents. We designed a fUS device for tethered brain imaging in freely moving rats based on a miniaturized ultrasound probe and a custom-made ultrasound scanner. We monitored CBV changes in rats during various behavioral states such as quiet rest, after whisker or visual stimulations, and in a food-reinforced operant task. We show that fUS imaging in freely moving rats could efficiently decode brain activity in real time.