Abstract
Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies.
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Acknowledgements
The project was funded by US National Institutes of Health (NIH) Contracts HHSN268201000044C and R01EB006432. This work was also supported by the National Research Foundation of Korea funded by the Korean Government (MSIP) 2012M3A6A3055694. A.D.A. was funded by the American Heart Association 14FTF20380185.
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Contributions
C.V., A.D.A., S.L. and R.W. designed the study, analyzed data and wrote the manuscript; C.V., A.D.A. and S.L. developed the system hardware and software and performed imaging experiments; A.D.A. performed surgical procedures.
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The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Fluoview interface.
Main user interface of the Fluoview program to control the FV1000 Olympus microscope. All acquisition parameters can be set from the user interface.
Supplementary Figure 2 Positioning of the heart stabilizer (magnified areas).
Magnified areas of Fig. 13. (a) Step 37. (b,c) Step 38. All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital and are in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
Supplementary Figure 3 Labview interface for retrospective gated acquisition.
Main user interface of the Labview program RetrospectiveGating.vi for retrospective gated acquisition.
Supplementary Figure 4 Labview interface for prospective gated acquisition.
Main user interface of the Labview program ProspectiveGating.vi for prospective gated acquisition.
Supplementary Figure 5 Comparison of native and paced electrocardiogram.
(a) The native ECG demonstrates P-waves due to atrial depolarization followed by the QRS complex of ventricular depolarization. (b) When paced, distinct pacing spikes from the stimulus isolator lead to capture of the ventricle and an altered QRS complex as well as loss of the conventional P-wave.
Supplementary Figure 6 Matlab interface for retrospective gated reconstructions.
Main interface of the custom-built GUI program RetrospectiveReconstruction for reconstructions obtained in retrospective gated acquisition mode.
Supplementary Figure 7 Matlab interface for prospective gated reconstructions.
Main interface of the custom-built GUI program ProspectiveReconstruction for reconstructions obtained in prospective gated acquisition mode.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1115 kb)
Supplementary Data 1
One zip file (Vinegoni-Software.zip) containing the Matlab and Labview software for prospective and retrospective acquisition and signal processing. (ZIP 5954 kb)
Supplementary Data 2
One zip file (Vinegoni-stabilizer.zip) containing the STEP and STL files for 3D printing and drawing of the stabilizer used in the protocol. (ZIP 33 kb)
Cable connections.
The movie illustrates the connections of the cable to the connector block NI BNC-2110 as indicated in Steps 5,6,7, and 9. (MP4 20098 kb)
ECG leads.
Insertion of the ECG leads into the paws' subcutaneous tissue (Step 16). The procedure is here performed for illustrative purposes on an euthanized animal. All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital and are in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. (MP4 5550 kb)
Imaging acquisition.
The movie illustrates a typical in vivo imaging acquisition session. Here test images are recorded to check for adequate image signal and to optimize the microscope settings (Step 45). All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital and are in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. (MP4 4737 kb)
Retrospective imaging acquisition.
The movie illustrates a sequence of consecutively acquired "raw" images (left panel) and the reconstructed stabilized images (right panel), in retrospective imaging acquisition modality. All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital and are in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. (MP4 4403 kb)
Prospective imaging acquisition.
The movie illustrates a sequence of consecutively acquired "raw" images (left panel) and the reconstructed stabilized images (right panel), in prospective imaging acquisition modality. All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital and are in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. (MP4 14017 kb)
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Vinegoni, C., Aguirre, A., Lee, S. et al. Imaging the beating heart in the mouse using intravital microscopy techniques. Nat Protoc 10, 1802–1819 (2015). https://doi.org/10.1038/nprot.2015.119
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DOI: https://doi.org/10.1038/nprot.2015.119
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