March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Label-free Morphometry of Retinal Nerve Fiber Bundles by Second-harmonic Generation Microscopy
Author Affiliations & Notes
  • Hyungsik Lim
    Physics, Hunter College of CUNY, New York, New York
  • John Danias
    Ophthalmology and Cell Biology, SUNY Downstate, Brooklyn, New York
  • Footnotes
    Commercial Relationships  Hyungsik Lim, None; John Danias, None
  • Footnotes
    Support  NIH Grant EY15224
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4985. doi:
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      Hyungsik Lim, John Danias; Label-free Morphometry of Retinal Nerve Fiber Bundles by Second-harmonic Generation Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4985.

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

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To demonstrate a novel label-free method to visualize and quantify the morphology of the retinal nerve fiber layer (RNFL) using nonlinear optical microscopy.


Simultaneous second-harmonic generation (SHG) microscopy and two-photon excitation fluorescence microscopy were performed to image the fresh ex-vivo rat retina. Retinal flat-mounts were placed in a flow chamber and perfused with physiological medium. The three-dimensional morphology of the RNFL was quantified in an unbiased manner using iterative intermeans algorithm for automatic thresholding. Time-lapse imaging of the retina was performed with SHG microscopy. To study the relationship between the structural integrity of microtubules (MTs) and the SHG signal, the retina was treated with a MT depolymerizing agent (nocodazole).


The optical properties, such as polarization- and spectral-dependence as well as beam directionality, confirmed that the intrinsic radiation from the axons of retinal ganglion cells (RGCs) is SHG. RNFL thickness, estimated from volumetric SHG images, ranged from 6 to 30µm. RNFL SHG signal did not significantly decline during 90 minutes in culture. In contrast when exposed to nocodazole SHG signal declined significantly (p<0.013, ANOVA) during the same time. The overall morphology of the RNFL was unaffected by depolymerization of MTs.


It has been suggested that disruptions in microtubule network are related to degeneration of RGCs, and that they occur earlier than RNFL thinning. SHG microscopy provides an alternative to interferometric methods used for the estimation of RNFL thickness, such as polarization-sensitive optical coherence tomography and scanning laser polarimetry. SHG has the theoretical advantage of higher specificity for imaging the microtubular network than birefringence-based methods.  

Keywords: imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • nerve fiber layer • microscopy: light/fluorescence/immunohistochemistry 

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