Three-dimensional motion tracking for high-resolution optical microscopy, in vivo
ABSTRACT 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.
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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 · 3.47 Impact Factor
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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 · 2.26 Impact Factor
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ABSTRACT: In conventional multi-probe fluorescence microscopy, narrow bandwidth filters on detectors are used to avoid bleed-through artefacts between probes. The limited bandwidth reduces the signal-to-noise ratio of the detection, often severely compromising one or more channels. Herein, we describe a process of using independent component analysis to discriminate the position of different probes using only a dichroic mirror to differentiate the signals directed to the detectors. Independent component analysis was particularly effective in samples where the spatial overlap between the probes is minimal, a very common case in cellular microscopy. This imaging scheme collects nearly all of the emitted light, significantly improving the image signal-to-noise ratio. In this study, we focused on the detection of two fluorescence probes used in vivo, NAD(P)H and ANEPPS. The optimal dichroic mirror cutoff frequency was determined with simulations using the probes spectral emissions. A quality factor, defined as the cross-channel contrast-to-noise ratio, was optimized to maximize signals while maintaining spatial discrimination between the probes after independent component analysis post-processing. Simulations indicate that a ∼3 fold increase in signal-to-noise ratio using the independent component analysis approach can be achieved over the conventional narrow-band filtering approach without loss of spatial discrimination. We confirmed this predicted performance from experimental imaging of NAD(P)H and ANEPPS in mouse skeletal muscle, in vivo. For many multi-probe studies, the increased sensitivity of this 'full bandwidth' approach will lead to improved image quality and/or reduced excitation power requirements.Journal of Microscopy 08/2014; 256(2). DOI:10.1111/jmi.12167 · 2.15 Impact Factor