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The image resolution and signal to noise is clearly improved in the tracked data set as implied from the cross-correlation data presented in Figure 4. To evaluate this approach during a physiological perturbation, we selected systemic hypoxia as it causes metabolic perturbations to the NADH redox state as well as significant changes in muscle volume and blood flow. For these experiments the reference image was collected and then a baseline was collected for two minutes.
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After this control period, the oxygen supply was replaced with nitrogen, and images were recorded for an additional three minutes. The procedure was repeated for seven independent trials.
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Figure 8 shows the X, Y, Z component of the motion compensation integral over time for each of the seven trials. The 3D correlation coefficient of the vascular structure over time was not collected or reported in these studies, as some of the degradation of the correlation is due to changes in the vascular volume.
The delay in a motion effect caused by the removal of oxygen was likely due to residual oxygen in the ventilation system, lungs, and blood.
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During the period before the oxygen is replaced with nitrogen to induce anoxia, the average rate of Z-axis motion drift across the seven trials was 6. After the anoxic event, the resting rate of drift was maintained for a period of approximately one minute, after which the rate of drift increased sharply to an average of In each of the seven trials, the total displacement in the Z-dimension exceeded the depth of the VOI 32 um.
This is an important observation since it confirms that the motion was greater than the FOV of the 3D volume, and therefore could not be corrected with post-processing methods alone. That is, the image planes of interest would not be in the 3D FOV at the beginning and end of the experiment without tracking.
Displacements Occurring in Hypoxia Trials. Each line represents a different animal. Time scale is in minutes with hypoxia was induced at time zero. Lag in effect is due to oxygen in the system. A Z displacement. B X Displacement. C Y Displacement. The displacements were consistent with the metabolic perturbations associated with hypoxia, including a large increase in NAD P H concentration.
As seen in the representative study presented in Figure 9 this system maintained good image resolution through the hypoxia transition and the NAD P H signal correlated well with the initiation of the spatial displacements associated with hypoxia. A positive displacement in the Z direction was observed in all trials See Figure 8a and was highly correlated with the changes in NADH P H, which likely represents a increase in muscle volume with hypoxia.
The tissue displacement in the X and Y directions was more random when not corrected for muscle axis as shown in Figure 8. Averaged combined image of red and blue channels and NAD P H correlated principal component of hypoxia with displacements for a single experiment.
A Image averaged over the entire experimental period revealing the maintenance of the imaging plane and resolution. The red and blue channels were averaged for this presentation with the blue channel be presented in green for better visualization. B For the same experiment as presented in A , the x, y and z displacement values and principal component amplitude that correlated with NAD P H fluorescence.
The principal component is normalized to the control period. Intra-vital optical microscopy image SNR and resolution is limited by tissue motion. In our experience using commercially available equipment, inherent in vivo tissue motion often dominates image quality more than the microscope optics, imaging probe, or excitation power applied as illustrated in Figure 7.
Thus, having a robust motion compensation system to permit image or spectral averaging is critical for in vivo optical microscopy studies. All images represent the average of the first volumes of each dataset. Images were auto window leveled individually. The top panels represents the red channel.