Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina

Robert J. Zawadzki, Pengfei Zhang, Azhar Zam, Eric B. Miller, Mayank Goswami, Xinlei Wang, Ravi S. Jonnal, Sang Hyuck Lee, Dae Yu Kim, John G. Flannery, John S. Werner, Marie E. Burns, Edward N. Pugh

Research output: Contribution to journalArticlepeer-review


Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a largescale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.

Original languageEnglish (US)
Pages (from-to)2191-2210
Number of pages20
JournalBiomedical Optics Express
Issue number6
StatePublished - May 21 2015


  • Active or adaptive optics
  • Imaging systems
  • Ophthalmic optics and devices
  • Ophthalmology
  • Optical coherence tomography
  • Visual optics, comparative animal models

ASJC Scopus subject areas

  • Biotechnology
  • Atomic and Molecular Physics, and Optics


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