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Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue.

Laffray S, Pagès S, Dufour H, De Koninck P, De Koninck Y, Côté D - PLoS ONE (2011)

Bottom Line: We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals.The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop.The performance of the system allows for efficient image locking even in conditions of random or irregular movements.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche Université Laval Robert-Giffard, Université Laval, Québec, Canada.

ABSTRACT
In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca(2+) imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.

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Working principle of the movement compensation device.(A) The illumination and collection paths for imaging (red trace) and movement compensation (orange trace) are independent. A feedback loop between the movement compensation device and a Z-axis piezo nanopositioner onto which the objective is mounted allows real-time Z movement compensation. Within the tissue, the Z position of the plane to be locked is chosen by adjusting the focal plane offset of the 1∶1 telescope. (B) The illumination for movement compensation is an off-axis hemicircular light beam. When the specimen is at the beam focus, the image of the beam is a point. The image of this point is reflected on the CCD detector as a bell-shaped intensity profile centred at a position corresponding to the in focus plane to be locked. When the plane is above (resp. below) focus, the image is a right-hand (resp. left-hand) hemicircle on the sample and the corresponding projection on the detector is a right (resp. left) shifted bell-shaped intensity profile. (C) Random movement imposed to a sample is compensated by an accurate mirror movement of the piezo onto which the objective is mounted. When the compensation system is activated, the relative movement between the two lies within 2.2 µm (RMS) (Difference). Displacements of the piezo and the specimen were independently calibrated.
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pone-0019928-g001: Working principle of the movement compensation device.(A) The illumination and collection paths for imaging (red trace) and movement compensation (orange trace) are independent. A feedback loop between the movement compensation device and a Z-axis piezo nanopositioner onto which the objective is mounted allows real-time Z movement compensation. Within the tissue, the Z position of the plane to be locked is chosen by adjusting the focal plane offset of the 1∶1 telescope. (B) The illumination for movement compensation is an off-axis hemicircular light beam. When the specimen is at the beam focus, the image of the beam is a point. The image of this point is reflected on the CCD detector as a bell-shaped intensity profile centred at a position corresponding to the in focus plane to be locked. When the plane is above (resp. below) focus, the image is a right-hand (resp. left-hand) hemicircle on the sample and the corresponding projection on the detector is a right (resp. left) shifted bell-shaped intensity profile. (C) Random movement imposed to a sample is compensated by an accurate mirror movement of the piezo onto which the objective is mounted. When the compensation system is activated, the relative movement between the two lies within 2.2 µm (RMS) (Difference). Displacements of the piezo and the specimen were independently calibrated.

Mentions: The device operation is based on the continuous optical monitoring of the position of the tissue and a feedback system to control the position of the objective to maintain constant the distance between the tissue and the objective (Fig. 1A), yielding effective movement compensation. This is achieved by illuminating the sample with an off-axis hemi-circular 785 nm light beam and by measuring the lateral displacement of the reflection onto a linear CCD detector as an indication of Z sample position (Fig. 1B). A control signal derived from the analysis of the reflected pattern is used to adjust a piezo nanopositioner onto which a water immersion objective is mounted, effectively closing the feedback loop (Fig. 1A). The optical paths for monitoring the position of the tissue and the one for imaging are independent (Fig. 1A). The Z position sampling operates at 1 kHz, but the feedback is effectively limited by the nanopositioner which can achieve 250 µm step displacements in 25 ms, which remains orders of magnitude faster than physiological fluctuations (e.g., heart rate and breathing).


Adaptive movement compensation for in vivo imaging of fast cellular dynamics within a moving tissue.

Laffray S, Pagès S, Dufour H, De Koninck P, De Koninck Y, Côté D - PLoS ONE (2011)

Working principle of the movement compensation device.(A) The illumination and collection paths for imaging (red trace) and movement compensation (orange trace) are independent. A feedback loop between the movement compensation device and a Z-axis piezo nanopositioner onto which the objective is mounted allows real-time Z movement compensation. Within the tissue, the Z position of the plane to be locked is chosen by adjusting the focal plane offset of the 1∶1 telescope. (B) The illumination for movement compensation is an off-axis hemicircular light beam. When the specimen is at the beam focus, the image of the beam is a point. The image of this point is reflected on the CCD detector as a bell-shaped intensity profile centred at a position corresponding to the in focus plane to be locked. When the plane is above (resp. below) focus, the image is a right-hand (resp. left-hand) hemicircle on the sample and the corresponding projection on the detector is a right (resp. left) shifted bell-shaped intensity profile. (C) Random movement imposed to a sample is compensated by an accurate mirror movement of the piezo onto which the objective is mounted. When the compensation system is activated, the relative movement between the two lies within 2.2 µm (RMS) (Difference). Displacements of the piezo and the specimen were independently calibrated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101223&req=5

pone-0019928-g001: Working principle of the movement compensation device.(A) The illumination and collection paths for imaging (red trace) and movement compensation (orange trace) are independent. A feedback loop between the movement compensation device and a Z-axis piezo nanopositioner onto which the objective is mounted allows real-time Z movement compensation. Within the tissue, the Z position of the plane to be locked is chosen by adjusting the focal plane offset of the 1∶1 telescope. (B) The illumination for movement compensation is an off-axis hemicircular light beam. When the specimen is at the beam focus, the image of the beam is a point. The image of this point is reflected on the CCD detector as a bell-shaped intensity profile centred at a position corresponding to the in focus plane to be locked. When the plane is above (resp. below) focus, the image is a right-hand (resp. left-hand) hemicircle on the sample and the corresponding projection on the detector is a right (resp. left) shifted bell-shaped intensity profile. (C) Random movement imposed to a sample is compensated by an accurate mirror movement of the piezo onto which the objective is mounted. When the compensation system is activated, the relative movement between the two lies within 2.2 µm (RMS) (Difference). Displacements of the piezo and the specimen were independently calibrated.
Mentions: The device operation is based on the continuous optical monitoring of the position of the tissue and a feedback system to control the position of the objective to maintain constant the distance between the tissue and the objective (Fig. 1A), yielding effective movement compensation. This is achieved by illuminating the sample with an off-axis hemi-circular 785 nm light beam and by measuring the lateral displacement of the reflection onto a linear CCD detector as an indication of Z sample position (Fig. 1B). A control signal derived from the analysis of the reflected pattern is used to adjust a piezo nanopositioner onto which a water immersion objective is mounted, effectively closing the feedback loop (Fig. 1A). The optical paths for monitoring the position of the tissue and the one for imaging are independent (Fig. 1A). The Z position sampling operates at 1 kHz, but the feedback is effectively limited by the nanopositioner which can achieve 250 µm step displacements in 25 ms, which remains orders of magnitude faster than physiological fluctuations (e.g., heart rate and breathing).

Bottom Line: We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals.The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop.The performance of the system allows for efficient image locking even in conditions of random or irregular movements.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche Université Laval Robert-Giffard, Université Laval, Québec, Canada.

ABSTRACT
In vivo non-linear optical microscopy has been essential to advance our knowledge of how intact biological systems work. It has been particularly enabling to decipher fast spatiotemporal cellular dynamics in neural networks. The power of the technique stems from its optical sectioning capability that in turn also limits its application to essentially immobile tissue. Only tissue not affected by movement or in which movement can be physically constrained can be imaged fast enough to conduct functional studies at high temporal resolution. Here, we show dynamic two-photon Ca(2+) imaging in the spinal cord of a living rat at millisecond time scale, free of motion artifacts using an optical stabilization system. We describe a fast, non-contact adaptive movement compensation approach, applicable to rough and weakly reflective surfaces, allowing real-time functional imaging from intrinsically moving tissue in live animals. The strategy involves enslaving the position of the microscope objective to that of the tissue surface in real-time through optical monitoring and a closed feedback loop. The performance of the system allows for efficient image locking even in conditions of random or irregular movements.

Show MeSH
Related in: MedlinePlus