May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Novel Techniques For Imaging Retinal Vessels Using Whole Retinas After Long Term Fixation
Author Affiliations & Notes
  • M.M. Campos
    Biological Imaging Core, National Eye Institute, Bethesda, MD
  • R.N. Fariss
    Biological Imaging Core, National Eye Institute, Bethesda, MD
  • W.G. Robison, Jr.
    Biological Imaging Core, National Eye Institute, Bethesda, MD
  • Footnotes
    Commercial Relationships  M.M. Campos, None; R.N. Fariss, None; W.G. Robison, Jr., None.
  • Footnotes
    Support  NON
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 2416. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      M.M. Campos, R.N. Fariss, W.G. Robison, Jr.; Novel Techniques For Imaging Retinal Vessels Using Whole Retinas After Long Term Fixation . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2416.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Abstract: : Purpose: Aldehyde–based fixatives (e.g., 4% paraformaldehyde in PBS, 10% buffered formalin, 2–4% buffered glutaraldehyde) provide a convenient and reliable method for preserving tissues for extend periods of time. A major disadvantage of long–term aldehyde fixation is background autofluorescence, a characteristic that increases with time. High levels of autofluorescence often complicate epifluorescence microscopy. This study utilized procedures that took advantage of the high levels of fixation–induced autofluorescence in intact human and rat retinas that had been stored in aldehyde fixatives for up to 17 years. Methods: Eyes were opened near the equator, the retina was carefully removed, the vitreous was brushed off, and the whole retina was mounted on a slide. A Leica SP2 laser scanning confocal microscope was utilized for precise characterization of the autofluorescence emission spectra and identification of the regions within these spectra that yielded optimal contrast between vascular and non–vascular tissues. Results: Collection of thin optical sections under conditions of optimal contrast, permitted high resolution imaging of vascular structures from "unstained" archival specimens. Fresh and lightly fixed retinas did not provide equivalent contrast or clear tissue distinction. Conclusions: These novel techniques have permitted, utilization of background autofluorescence to visualize minute structural features of retinal vessels, identification and quantification of normal and pathological vascular features in unstained archival samples, preservation of complex 3D vascular networks and localization of vascular lesions to specific retinal layers.Thus, the increased autofluorescence arising from long–term fixation that would otherwise cause unwanted background signal became an asset.

Keywords: image processing • retina • microscopy: fixation processing 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×