title: Compact non-contact total emission detection for in vivo multiphoton excitation microscopy.
abstract: We describe a compact, non-contact design for a total emission detection (c-TED) system for intra-vital multiphoton imaging. To conform to a standard upright two-photon microscope design, this system uses a parabolic mirror surrounding a standard microscope objective in concert with an optical path that does not interfere with normal microscope operation. The non-contact design of this device allows for maximal light collection without disrupting the physiology of the specimen being examined. Tests were conducted on exposed tissues in live animals to examine the emission collection enhancement of the c-TED device compared to heavily optimized objective-based emission collection. The best light collection enhancement was seen from murine fat (5×-2× gains as a function of depth), whereas murine skeletal muscle and rat kidney showed gains of over two and just under twofold near the surface, respectively. Gains decreased with imaging depth (particularly in the kidney). Zebrafish imaging on a reflective substrate showed close to a twofold gain throughout the entire volume of an intact embryo (approximately 150 μm deep). Direct measurement of bleaching rates confirmed that the lower laser powers, enabled by greater light collection efficiency, yielded reduced photobleaching in vivo. The potential benefits of increased light collection in terms of speed of imaging and reduced photo-damage, as well as the applicability of this device to other multiphoton imaging methods is discussed.
authors.affiliation: NHLBI Light Microscopy Facility, National Institutes of Health, Bethesda, Maryland 20892-1061.
authors.forename: Christian A
authors.initials: CA
authors.lastname: Combs
authors.affiliation: NHLBI Laboratory of Molecular Biophysics, National Institutes of Health, Bethesda, Maryland 20892-1061.
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Smirnov
authors.affiliation: NHLBI Laboratory of Cardiac Energetics, National Institutes of Health, Bethesda, Maryland 20892-1061.
authors.forename: Brian
authors.initials: B
authors.lastname: Glancy
authors.affiliation: NICHD Section on Biomedical Stochastic Physics, National Institutes of Health, Bethesda, Maryland 20892-1061.
authors.forename: Nader S
authors.initials: NS
authors.lastname: Karamzadeh
authors.affiliation: NICHD Section on Biomedical Stochastic Physics, National Institutes of Health, Bethesda, Maryland 20892-1061.
authors.forename: Amir H
authors.initials: AH
authors.lastname: Gandjbakhche
authors.affiliation: Intelligent Imaging Innovations, Inc., Denver, CO 80216.
authors.forename: Glen
authors.initials: G
authors.lastname: Redford
authors.affiliation: Intelligent Imaging Innovations, Inc., Denver, CO 80216.
authors.forename: Karl
authors.initials: K
authors.lastname: Kilborn
authors.affiliation: NHLBI Laboratory of Molecular Biophysics, National Institutes of Health, Bethesda, Maryland 20892-1061.
authors.forename: Jay R
authors.initials: JR
authors.lastname: Knutson
authors.affiliation: NHLBI Laboratory of Cardiac Energetics, National Institutes of Health, Bethesda, Maryland 20892-1061.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Balaban
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000033:anatomy & histology; D007091:Image Processing, Computer-Assisted; D007668:Kidney / Q000033:anatomy & histology; D007834:Lasers; D008055:Lipids / Q000032:analysis; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D018482:Muscle, Skeletal / Q000033:anatomy & histology; D038761:Photobleaching; D051381:Rats; D017207:Rats, Sprague-Dawley; D059629:Signal-To-Noise Ratio; D015027:Zebrafish / Q000033:anatomy & histology
chemical_list: D008055:Lipids
keywords: Imaging; light collection; two-photon microscopy