title: Deep tissue fluorescence imaging and in vivo  biological applications.
abstract: We describe a novel technical approach with enhanced fluorescence detection capabilities in twophoton microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.
authors.affiliation: University of California at Irvine, Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, Irvine, California 92697, USA.
authors.forename: Viera
authors.initials: V
authors.lastname: Crosignani
authors.forename: Alexander
authors.initials: A
authors.lastname: Dvornikov
authors.forename: Jose S
authors.initials: JS
authors.lastname: Aguilar
authors.forename: Chiara
authors.initials: C
authors.lastname: Stringari
authors.forename: Robert
authors.initials: R
authors.lastname: Edwards
authors.forename: William W
authors.initials: WW
authors.lastname: Mantulin
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
mesh_terms: D000818:Animals; D003106:Colon / Q000033:anatomy & histology; D003952:Diagnostic Imaging / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007421:Intestine, Small / Q000033:anatomy & histology; D051379:Mice; D008822:Mice, Transgenic; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D055620:Optical Phenomena; D019047:Phantoms, Imaging
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins