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: Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.AJP Cell Physiology 10/2002; 283(3):C905-16. · 3.71 Impact Factor
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ABSTRACT: Capillary perfusion in the brain is characterized by an essentially continuous flow of erythrocytes and plasma in almost all capillaries. Rapid fluctuations and spatial heterogeneity or red blood cell (RBC) velocity (0.5-1.8 mm/s) within the capillary network are present. In addition, low-frequency (4-8 cpm) synchronous oscillations in RBC velocity in the capillary network emerge when perfusion to cerebral tissue is challenged. Despite the tortuous, three-dimensional architecture of microvessels, functional intercapillary anastomoses are absent. At rest, red cells travel through the capillary network in 100-300 ms along 150- to 500-micron-long paths. Physiological challenges elicit sizable changes in RBC velocity with a minor role for capillary recruitment, change in capillary diameter, or flow shunting. During acute hypoxia, RBC velocity increases in all capillaries; the corresponding response to hypereapnia is more complex and involves redistribution of capillary flow toward more homogeneous perfusion. The response of capillary flow to decreased perfusion pressure reflects autoregulation of cerebral blood flow but also involves intranetwork redistribution of RBC flow between two populations of capillaries, postulated as thoroughfare channels and exchange capillaries. Flow reserve may be provided by the thoroughfare channels and may help maintain flow velocity and capillary exchange and protect the microcirculation from perfusion failure. Isovolemic hemodilution increases RBC velocity three- to fourfold and increases RBC flux to a moderate degree with a relatively small decrease in capillary hematocrit, under normal and compromised arterial blood supply. In cerebral ischemia, leukocyte adhesion is enhanced and appears reversible when the ischemia is moderate but may be progressive when the injury is severe. The observed flow behavior suggests the presence of a physiological regulatory mechanism of cerebral capillary flow that may involve communication among various microvascular and parenchymal cells and utilize locally acting endothelial and parenchymal mediators such as endothelium-derived relaxing factor or nitric oxide.Microcirculation 07/1997; 4(2):233-52. · 2.76 Impact Factor
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ABSTRACT: This study investigated the relationship between hypoxia and the rate of fatigue development in contracting rat hindlimb muscles composed primarily of different fibre types. Hindlimb muscles of 11 rats were exposed, and the soleus (SOL) and gastrocnemius/plantaris (GP) were each isolated with circulation intact and attached to individual force transducers. Rats were then equilibrated with either normoxic (N; arterial partial pressure of O(2) 87.7 +/- 1.5 mmHg) or hypoxic conditions (H; arterial partial pressure of O(2) 30.0 +/- 2.4 mmHg) using an inspired O(2) fraction of 0.21 and 0.10, respectively. The stimulation protocol consisted of 2 min each at 0.125, 0.25, 0.33 and 0.5 tetanic contractions s(-1) sequentially for both conditions. Following the 8 min stimulation period, relative developed muscle tension (% of maximal) was nearly identical for both H and N in SOL (54.2 +/- 3.5 versus 54.3 +/- 4.2%), but was significantly (P < 0.05) lower in H than N (10.8 +/- 0.9 versus 43.0 +/- 8.9%) in GP, indicating a greater amount of fatigue during hypoxia only in the GP. Soleus phosphocreatine (PCr) content fell to similar levels (24.1 +/- 1.6 versus 21.1 +/- 4.9 mmol (kg dry weight (dw))(-1)) during both H and N, but in the white portion of the gastrocnemius (WG), PCr was significantly lower following H than N (14.3 +/- 1.5 versus 34.0 +/- 6.0 mmol (kg dw)(-1)). Similarly, muscle lactate increased in both fibre types at fatigue, but only in WG was the increase significantly greater with H (SOL 7.1 +/- 2.0 versus 5.3 +/- 1.1 mmol (kg dw)(-1); WG 13.7 +/- 4.5 versus 5.3 +/- 2.2 mmol (kg dw)(-1)). Increases in calculated muscle [H(+)], free ADP and free AMP were similar between N and H in SOL but were significantly greater during H compared with N in WG. These data demonstrate that hypoxia induces greater fatigue and disruption of cellular homeostasis in rat hindlimb muscle composed primarily of fibres with low oxidative capacity compared with those of a more oxidative type.Experimental Physiology 10/2007; 92(5):887-94. · 2.79 Impact Factor