All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single-and two-photon near-infrared excitation microscopes

Pinaki Sarder, Siavash Yazdanfar, Walter J. Akers, Rui Tang, Gail P. Sudlow, Christopher Egbulefu, Samuel Achilefu

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

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 μ m. The NIR/NIR MPM enabled greater than fivefold imaging depth (500 μ m) 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.

Original languageEnglish
Article number106012
JournalJournal of biomedical optics
Volume18
Issue number10
DOIs
StatePublished - Oct 2013

Keywords

  • all-near-infrared multiphoton microscopy
  • cyanine dyes
  • laser-induced photodamage
  • multiphoton microscopy
  • near-infrared fluorescent molecular probes
  • near-infrared single-photon confocal microscopy

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