Improved intravital microscopy via synchronization of respiration and holder stabilization

Sungon Lee; Claudio Vinegoni; Ralph Weissleder; Paolo F. Feruglio

doi:10.1117/1.JBO.17.9.096018

21 September 2012 Improved intravital microscopy via synchronization of respiration and holder stabilization

Sungon Lee, Claudio Vinegoni, Ralph Weissleder, Paolo F. Feruglio

Author Affiliations +

Funded by: National Institutes of Health, Department of Health and Human Services, Institute of Biomedical Engineering

Journal of Biomedical Optics, Vol. 17, Issue 9, 096018 (September 2012). https://doi.org/10.1117/1.JBO.17.9.096018

Abstract

A major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy.

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Sungon Lee, Claudio Vinegoni, Ralph Weissleder, and Paolo F. Feruglio "Improved intravital microscopy via synchronization of respiration and holder stabilization," Journal of Biomedical Optics 17(9), 096018 (21 September 2012). https://doi.org/10.1117/1.JBO.17.9.096018

Published: 21 September 2012

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