title: Improved intravital microscopy via synchronization of respiration and holder stabilization.
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.
authors.affiliation: Massachusetts General Hospital and Harvard Medical School, Center for System Biology, Richard B. Simches Research Center, 185 Cambridge Street, Boston, Massachusetts 02114, USA.
authors.forename: Sungon
authors.initials: S
authors.lastname: Lee
authors.forename: Claudio
authors.initials: C
authors.lastname: Vinegoni
authors.forename: Paolo Fumene
authors.initials: PF
authors.lastname: Feruglio
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
mesh_terms: D017076:Computer-Aided Design; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D007103:Immobilization / Q000295:instrumentation*; D018613:Microscopy, Confocal / Q000295:instrumentation*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D015203:Reproducibility of Results; D055425:Respiratory-Gated Imaging Techniques / Q000295:instrumentation*; D012680:Sensitivity and Specificity; D013382:Subtraction Technique / Q000295:instrumentation*