July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Label-Free Imaging of Bipolar Cell Axons in Fresh Retina by Second-Harmonic Generation
Author Affiliations & Notes
  • Hyungsik Lim
    Physics, Hunter College of CUNY, New York, New York, United States
  • Festa Bucinca-Cupallari
    Physics, Hunter College of CUNY, New York, New York, United States
  • Footnotes
    Commercial Relationships   Hyungsik Lim, None; Festa Bucinca-Cupallari, None
  • Footnotes
    Support  NIH Grant GM121198
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 6093. doi:
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      Hyungsik Lim, Festa Bucinca-Cupallari; Label-Free Imaging of Bipolar Cell Axons in Fresh Retina by Second-Harmonic Generation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6093.

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

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Purpose : The bipolar cells (BCs) are the primary neuron in the retina relaying visual information from the photoreceptors to the retinal ganglion cells (RGCs), and their axons comprise the inner plexiform layer (IPL) connecting the inner and outer retinas{Euler, 2014 #7476}. Here a novel, label-free method is presented for visualization of the BC axons in the fresh retina ex vivo. Its utility is demonstrated for testing the persistence of BC axons in the course of glaucoma, despite the synaptic loss and dendritic atrophy of the RGCs known to occur in the process{Williams, 2013 #7477;Berry, 2015 #7473;Berry, 2015 #7473}.

Methods : Second-harmonic generation (SHG) microscopy was performed for imaging the BC axons in the fresh mouse retinas. A SHG setup was employed similarly as we have previously used for imaging the RGC axons{Lim, 2012 #6701;Sharoukhov, 2018 #7470}. The fresh retinal flatmounts were prepared for imaging. Exploiting the broadband excitability of SHG, wavelengths of 800 and 1200 nm were compared. To confirm that the origin of non-RGC SHG signal was indeed the BC axons, transgenic Thy1-YFP mice expressing yellow fluorescent protein (YFP) in BCs were imaged by simultaneous two-photon excited fluorescence (TPEF) and SHG microscopy. To test a hypothesis relevant to glaucoma, i.e., that the BC axons persist during glaucomatous pathogenesis, DBA/2J mice were used as an experimental model of glaucoma and DBA/2J-Gpnmb+ as a non-glaucomatous control.

Results : SHG signals arising from uniformly polarized microtubules (MTs) were acquired from the BC axons in the IPL as well as the RGC axons. The length of BC axons was approximately 30 µm. Due to the axial orientation, the BC axons did not respond much differently for two orthogonal polarizations of the excitation beam, unlike the RGC axons. The near-IR (NIR) excitation at 1200 nm for SHG imaging was safer to the retina and the quality of SHG images obtained at 1200 nm was comparable to or exceeded those at 800 nm. The co-localization of SHG signal with Thy1-YFP showed the spatial relationship of the BC axons with other cellular features in the IPL. Finally, SHG images of the retinas of DBA/2J mice (>12 months old) showed the BC axons persistent in regions where the RGC axons were lost.

Conclusions : SHG microscopy is a novel label-free technique for visualizing the BC axons, by which the roles in pathology and visual processing can be investigated.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.




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