title: Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging.
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.
authors.affiliation: Department of Anatomy and Structural Biology, and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: David
authors.initials: D
authors.lastname: Entenberg
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Wyckoff
authors.forename: Bojana
authors.initials: B
authors.lastname: Gligorijevic
authors.forename: Evanthia T
authors.initials: ET
authors.lastname: Roussos
authors.forename: Vladislav V
authors.initials: VV
authors.lastname: Verkhusha
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Pollard
authors.forename: John
authors.initials: J
authors.lastname: Condeelis
mesh_terms: D000818:Animals; D005260:Female; D007091:Image Processing, Computer-Assisted; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D012984:Software