title: All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes.
abstract: The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100 μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500 μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms.
authors.affiliation: Washington University, Department of Radiology, 4225 Scott Avenue, St. Louis, Missouri 63108.
authors.forename: Pinaki
authors.initials: P
authors.lastname: Sarder
authors.forename: Siavash
authors.initials: S
authors.lastname: Yazdanfar
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Akers
authors.forename: Rui
authors.initials: R
authors.lastname: Tang
authors.forename: Gail P
authors.initials: GP
authors.lastname: Sudlow
authors.forename: Christopher
authors.initials: C
authors.lastname: Egbulefu
authors.forename: Samuel
authors.initials: S
authors.lastname: Achilefu
mesh_terms: D000818:Animals; D002232:Carbocyanines; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003287:Contrast Media / Q000737:chemistry* / Q000494:pharmacology; D004655:Emulsions; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000494:pharmacology; D006651:Histocytochemistry; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007668:Kidney / Q000737:chemistry / Q000528:radiation effects; D007834:Lasers; D008297:Male; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D019047:Phantoms, Imaging; D010743:Phospholipids; D013024:Soybean Oil; D019265:Spectroscopy, Near-Infrared / Q000379:methods*
chemical_list: D002232:Carbocyanines; D003287:Contrast Media; D004655:Emulsions; D005456:Fluorescent Dyes; D010743:Phospholipids; C089187:cyanine dye 3; C545823:soybean oil, phospholipid emulsion; D013024:Soybean Oil