When conducting optical imaging experiments, in vivo, the signal to noise ratio and effective spatial and temporal resolution is fundamentally limited by physiological motion of the tissue. A three-dimensional (3D) motion tracking scheme, using a multiphoton excitation microscope with a resonant galvanometer, (512 × 512 pixels at 33 frames s(-1)) is described to overcome physiological motion, in vivo. The use of commercially available graphical processing units permitted the rapid 3D cross-correlation of sequential volumes to detect displacements and adjust tissue position to track motions in near real-time. Motion phantom tests maintained micron resolution with displacement velocities of up to 200 μm min(-1), well within the drift observed in many biological tissues under physiologically relevant conditions. In vivo experiments on mouse skeletal muscle using the capillary vasculature with luminal dye as a displacement reference revealed an effective and robust method of tracking tissue motion to enable (1) signal averaging over time without compromising resolution, and (2) tracking of cellular regions during a physiological perturbation.
"ization for all phases of the cardiac cycle is through active motion compensation . In this method the relative residual motion present between the imaging device ( i . e . , objective ) and the imaged tissue can be actively canceled by tracking in real time the tissue position ( Nakamura et al . , 2001 ; Lee et al . , 2008 ; Yuen et al . , 2009 ; Bakalar et al . , 2012 ) and accordingly shifting the objective . This cancelation of relative movement virtually freezes in space both the objective and imaged sample leading to motion artifact free images ."
[Show abstract][Hide abstract] ABSTRACT: Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases.
Frontiers in Physiology 05/2015; 6. DOI:10.3389/fphys.2015.00147 · 3.53 Impact Factor
"It is therefore recommended, whenever possible, to adopt passive mechanical stabilizers in combination with their use in a synergistic effort aimed at reducing the total amount of motion , . Alternatively motion compensation along the plane perpendicular to the optical axis has been demonstrated by controlling an XY translational stage  while Z motion compensation has also been achieved using an objective focus motor  "
[Show abstract][Hide abstract] ABSTRACT: Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions.
IEEE Journal of Selected Topics in Quantum Electronics 03/2014; 20(2). DOI:10.1109/JSTQE.2013.2279314 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective
To provide insight into mitochondrial function in vivo, we evaluated the 3D spatial relationship between capillaries, mitochondria, and muscle fibers in live mice. Methods3D volumes of in vivo murine Tibialis anterior muscles were imaged by multi-photon microscopy (MPM). Muscle fiber type, mitochondrial distribution, number of capillaries, and capillary-to-fiber contact were assessed. The role of myoglobin-facilitated diffusion was examined in myoglobin knockout mice. Distribution of GLUT4 was also evaluated in the context of the capillary and mitochondrial network. ResultsMPM revealed that 43.6 ± 3.3% of oxidative fiber capillaries had ≥ 50% of their circumference embedded in a groove in the sarcolemma, in vivo. Embedded capillaries were tightly associated with dense mitochondrial populations lateral to capillary grooves and nearly absent below the groove. Mitochondrial distribution, number of embedded capillaries, and capillary-to-fiber contact were proportional to fiber oxidative capacity and unaffected by myoglobin knockout. GLUT4 did not preferentially localize to embedded capillaries. Conclusions
Embedding capillaries in the sarcolemma may provide a regulatory mechanism to optimize delivery of oxygen to heterogeneous groups of muscle fibers. We hypothesize that mitochondria locate to paravascular regions due to myofibril voids created by embedded capillaries, not to enhance the delivery of oxygen to the mitochondria.This article is protected by copyright. All rights reserved.
Microcirculation (New York, N.Y.: 1994) 10/2013; 21(2). DOI:10.1111/micc.12098 · 2.57 Impact Factor
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