Imaging the beating heart in the mouse using intravital microscopy techniques (original) (raw)

Nature Protocols volume 10, pages 1802–1819 (2015)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 12 print issues and online access

$259.00 per year

only $21.58 per issue

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Additional access options:

Similar content being viewed by others

References

  1. Shih, A.Y. et al. Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain. J. Cereb. Blood Flow Metab. 32, 1277–1309 (2012).
    Article CAS Google Scholar
  2. Devor, A. et al. Frontiers in optical imaging of cerebral blood flow and metabolism. J. Cereb. Blood Flow Metab. 32, 1259–1276 (2012).
    Article CAS Google Scholar
  3. Murooka, T.T. et al. HIV-infected T cells are migratory vehicles for viral dissemination. Nature 490, 283–287 (2012).
    Article CAS Google Scholar
  4. Sung, J.H. et al. Chemokine guidance of central memory T cells is critical for antiviral recall responses in lymph nodes. Cell 150, 1249–1263 (2012).
    Article CAS Google Scholar
  5. Pittet, M.J. & Weissleder, R. Intravital imaging. Cell 147, 983–991 (2011).
    Article CAS Google Scholar
  6. Condeelis, J. & Weissleder, R. In vivo imaging in cancer. Cold Spring Harb. Perspect. Biol. 2, a003848 (2010).
    Article CAS Google Scholar
  7. Fukumura, D., Duda, D.G., Munn, L.L. & Jain, R.K. Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models. Microcirculation 17, 206–225 (2010).
    Article CAS Google Scholar
  8. Rubart, M., Wang, E., Dunn, K.W. & Field, L.J. Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts. Am. J. Physiol. Cell Physiol. 284, C1654–C1668 (2003).
    Article CAS Google Scholar
  9. Matsumoto-Ida, M., Akao, M., Takeda, T., Kato, M. & Kita, T. Real-time 2-photon imaging of mitochondrial function in perfused rat hearts subjected to ischemia/reperfusion. Circulation 114, 1497–1503 (2006).
    Article CAS Google Scholar
  10. Bub, G. et al. Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro. Am. J. Physiol. Heart Circ. Physiol. 298, H1616–H1625 (2010).
    Article CAS Google Scholar
  11. Lee, S. et al. Real-time in vivo imaging of the beating mouse heart at microscopic resolution. Nat. Commun. 3, 1054 (2012).
    Article Google Scholar
  12. Aguirre, A.D., Vinegoni, C., Sebas, M. & Weissleder, R. Intravital imaging of cardiac function at the single-cell level. Proc. Natl. Acad. Sci. USA 111, 11257–11262 (2014).
    Article CAS Google Scholar
  13. Li, W. et al. Intravital 2-photon imaging of leukocyte trafficking in beating heart. J. Clin. Invest. 122, 2499–2508 (2012).
    Article CAS Google Scholar
  14. Jung, K. et al. Endoscopic time-lapse imaging of immune cells in infarcted mouse hearts. Circ. Res. 112, 891–899 (2013).
    Article CAS Google Scholar
  15. Vinegoni, C., Lee, S., Gorbatov, R. & Weissleder, R. Motion compensation using a suctioning stabilizer for intravital microscopy. Intravital 1, 115–121 (2012).
    Article Google Scholar
  16. Scott, A.D., Keegan, J. & Firmin, D.N. Motion in cardiovascular MR imaging. Radiology 250, 331–351 (2009).
    Article Google Scholar
  17. Tsao, J. & Kozerke, S. MRI temporal acceleration techniques. J. Magn. Reson. Imaging 36, 543–560 (2012).
    Article Google Scholar
  18. Atkinson, D.J. & Edelman, R.R. Cineangiography of the heart in a single breath hold with a segmented turboFLASH sequence. Radiology 178, 357–360 (1991).
    Article CAS Google Scholar
  19. Lee, S., Vinegoni, C., Feruglio, P.F. & Weissleder, R. Improved intravital microscopy via synchronization of respiration and holder stabilization. J. Biomed. Opt. 17, 96018 (2012).
    PubMed Google Scholar
  20. Vinegoni, C. et al. Sequential average segmented microscopy for high signal-to-noise ratio motion-artifact-free in vivo heart imaging. Biomed. Opt. Express 4, 2095–2106 (2013).
    Article Google Scholar
  21. Constantinides, C., Mean, R. & Janssen, B.J. Effects of isoflurane anesthesia on the cardiovascular function of the C57BL/6 mouse. ILAR J. 52, e21–e31 (2011).
    PubMed Google Scholar
  22. Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S. & Kass, D.A. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat. Protoc. 3, 1422–1434 (2008).
    Article CAS Google Scholar
  23. Tarnavski, O. et al. Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies. Physiol. Genomics 16, 349–360 (2004).
    Article CAS Google Scholar
  24. Redel, A. et al. Impact of ischemia and reperfusion times on myocardial infarct size in mice in vivo. Exp. Biol. Med. 233, 84–93 (2008).
    Article CAS Google Scholar
  25. Ho, D. et al. Heart rate and electrocardiography monitoring in mice. Curr. Protoc. Mouse Biol. 1, 123–139 (2011).
    Article CAS Google Scholar

Download references

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.

Author information

Author notes

  1. Claudio Vinegoni and Aaron D Aguirre: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA
    Claudio Vinegoni, Aaron D Aguirre, Sungon Lee & Ralph Weissleder
  2. Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
    Aaron D Aguirre
  3. School of Electrical Engineering, Hanyang University, Ansan, Republic of Korea
    Sungon Lee

Authors

  1. Claudio Vinegoni
    You can also search for this author inPubMed Google Scholar
  2. Aaron D Aguirre
    You can also search for this author inPubMed Google Scholar
  3. Sungon Lee
    You can also search for this author inPubMed Google Scholar
  4. Ralph Weissleder
    You can also search for this author inPubMed Google Scholar

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.

Corresponding author

Correspondence toClaudio Vinegoni.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

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)

Rights and permissions

About this article

Cite this article

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

Download citation

This article is cited by

Associated content