KEYWORDS: Calcium, Hemodynamics, Two photon imaging, Neurons, In vivo imaging, Red blood cells, Two photon excitation microscopy, Blood vessels, Video, Biological imaging
SignificanceThe studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device.AimWe introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400 kHz and a maximum frame rate of 10,000 frames per second (fps) at 250 × 40 pixels. For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning.ApproachWith this combinatorial device that supports both large-view navigation and small-view high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs).ResultsWe measured dendritic calcium propagation velocity (80 / 62.5 − 116.7 μm / ms) in OGB-1-labeled single cortical neurons in mice in vivo. To benchmark the spatial precision and detection sensitivity of measurement in vivo, we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille’s law of laminar flow.ConclusionsThis proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo.
KEYWORDS: Microelectromechanical systems, Mirrors, Luminescence, Micromirrors, Photodynamic therapy, In vivo imaging, Prototyping, Tumors, Tissues, Surgery
Nowadays, the ability to diagnose brain tumors intraoperatively and to delineate their margins, as accurately as possible, is of primordial importance during surgery. However, the exact tumor boundaries and the infiltrated tumor cells outside its solid volume are difficult to be found by the surgeon due to the similar visual appearances especially at the margins, leading in many cases to poor surgical outcomes and a high risk of recurrences. To solve this issue, our group develop a two-photon multimodal endomicroscope to analyze the endogenous fluorescence of cerebral tissues in order to provide reliable informations intraoperatively on the tissues nature at cellular level. For that, and in order to implement the imaging modality in our endomicroscope, we launched a study based on an original miniaturized scanning system to be coupled with our endomicroscope. In this work, we characterize a scanning system prototype based on an electrothermally-actuated scanning micro-mirror. This type of scanning devices can provide a wide linear scan range at a low driving voltage and a high stable scanning speed along a straight scan line which help to acquire high-quality images.
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