Protocol | Published:

Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging

Nature Protocols volume 6, pages 15001520 (2011) | Download Citation

Abstract

Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via 'over-clocking' of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in 24 h.

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Acknowledgements

This work was supported by grants to J.C. from the US National Institutes of Health (NCI100324), the National Cancer Institute's Tumor Microenvironment Network, the Gruss Lipper Biophotonics Center and Mouse Models of Human Cancers Consortium; and grants to V.V.V. from the US National Institutes of Health (GM073913). B.G. was supported by a Charles H. Revson fellowship. We thank M. Metz, member of the Gruss Lipper Biophotonics Center, for his help with design and development. We also thank the members of the V.V.V. lab for useful discussions and M. Roh-Johnson for preparing the in vitro cell cultures.

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Affiliations

  1. Department of Anatomy and Structural Biology, and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.

    • David Entenberg
    • , Jeffrey Wyckoff
    • , Bojana Gligorijevic
    • , Evanthia T Roussos
    • , Vladislav V Verkhusha
    • , Jeffrey W Pollard
    •  & John Condeelis

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Contributions

D.E., J.W. and J.C. designed the microscope and plug-in. D.E. built the microscope and wrote the plug-in. B.G. transfected proteins into cells and grew the mouse tumors. E.T.R. provided the ROI_Tracker analysis data. V.V.V. developed the TagRFP657 protein and its stably expressing MTLn3 tumor cell line. J.W.P., J.W. and J.C. developed the transgenic Dendra2 mouse model. D.E., J.W., B.G. and J.C. wrote the paper. J.C. defined the microscope performance characteristics required to address the biological application, and overall design was done by D.E. and J.C.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to John Condeelis.

Supplementary information

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    Supplementary Figure 1

    3D Cad of Multiphoton Setup

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DOI

https://doi.org/10.1038/nprot.2011.376

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