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

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

Research output: Contribution to journalArticle

62 Scopus citations

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 24h.

Original languageEnglish (US)
Pages (from-to)1500-1520
Number of pages21
JournalNature Protocols
Volume6
Issue number10
DOIs
StatePublished - Oct 1 2011

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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