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

Nature Protocols volume 6, pages 1500–1520 (2011)Cite this article

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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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Figure 1: Optical layout of custom-built TLMPM.
Figure 2: Laser wavelengths and spectra of commonly used combinations of fluorescent proteins with the TLMPM.
Figure 3: Optical layout detail of multiphoton functional unit A—Tsunami beam path.
Figure 4: Optical layout detail of multiphoton functional unit B—Mai Tai–Mai Tai pump beam path.
Figure 5: Optical layout detail of multiphoton functional unit C—Mai Tai–Opal beam path.
Figure 6: Optical layout detail of multiphoton functional unit D—combining optics and scanning optics.
Figure 7: Optical layout detail of multiphoton functional unit E—microscope and detector box.
Figure 8: Effect of GVD compensator.
Figure 9: Electronic schematic of blanking circuit.
Figure 10: Electronic schematic of two-laser multiphoton microscope.
Figure 11: Internal connections for generating timing signals and clocking acquisition.
Figure 12: Over-clocked acquisition of PMT signals.
Figure 13: The ROI tracker user interface.
Figure 14: An in vitro demonstration of color subtraction technique.
Figure 15: In vivo images of exogenously grown tumor in immunodeficient mouse.
Figure 16: Comparison between traditional and over-clocked acquisition.
Figure 17: Use of the two-laser multiphoton microscope to study dissemination.
Figure 18: ROI _Tracker analysis.
Figure 19: ROI _Tracker analysis.
Figure 20: Tracking ability in ROI_Tracker Software.

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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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Authors and Affiliations

  1. Department of Anatomy and Structural Biology, and Gruss Lipper Biophotonics Center,

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

  2. , 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.

Corresponding author

Correspondence to John Condeelis.

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Supplementary information

Supplementary Figure 1

3D Cad of Multiphoton Setup (PDF 18261 kb)

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Entenberg, D., Wyckoff, J., Gligorijevic, B. et al. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat Protoc 6, 1500–1520 (2011). https://doi.org/10.1038/nprot.2011.376

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