Abstract
Intravital multiphoton microscopy has become one of the central tools in the investigation of dynamic cellular activity and function in living animals under nearly physiological conditions and is particularly important for studying the dynamic immune system. During intravital imaging in mice, periodic motion of tissue caused by respiration induces significant shifts of the imaged region. In slow laser scanning imaging modalities such as multiphoton microscopy this movement can lead to considerable distortion and discontinuity in 3D of the acquired images. Here, we introduce VivoFollow 2, a toolkit that concurrent with image acquisition performs a precise measurement of the respective image distortion enabling subsequent automated correction of the imaging data. Recovery of one 3D image stack, corresponding to the tomographic tissue sectioning by the optical plane from each single raw image stack, preserves the time continuity within each image stack. Implementation of VivoFollow 2 thus enables a minimization of motion artifacts in tissues exposed to periodic movements and allows for long term time-lapse imaging and subsequent precise image analysis of the dynamics of cellular and humoral factors in vivo.
Highlights
In the last two decades, intravital multiphoton microscopy has become one of the central tools in basic and preclinical biological research
We have developed the VivoFollow 2 system, which allows for the measurement and correction of distortion caused by periodic motion, such as animal breathing activity during relatively slow 2-photon intravital microscopy (2P-IVM) image acquisition
While previously described methods discard distorted frames [24] or recover undistorted images by combining tiles of multiple image stacks using pattern matching [25] or gating schemes [26], our method recovers one image stack corresponding to the optical plane tomographic tissue sectioning from each single raw image stack
Summary
In the last two decades, intravital multiphoton microscopy has become one of the central tools in basic and preclinical biological research. It allows for the investigation of dynamic cellular activity and function in living animals under nearly physiological conditions (reviewed in [1] and [2]). 2P-IVM has been used to visualize a wide variety of biological processes, such as the homing and interaction of dendritic cells with T cells in lymph nodes [4, 5], the migration of T cells across the blood-brain barrier into the central nervous system (CNS) [6,7,8], the processes of tumor angiogenesis and metastasis (reviewed in [9] and [10]), and the post-arrest behavior of immune cells on endothelial cells lining blood vessels during their extravasation.
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