September 2016
Volume 57, Issue 12
ARVO Annual Meeting Abstract  |   September 2016
Low-power two-photon fluorescence imaging of mouse retinal pigmented epithelium in vivo
Author Affiliations & Notes
  • Nathan Alexander
    Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States
  • Patrycjusz Stremplewski
    Nicolaus Copernicus University, Torun, Poland
  • Maciej D Wojtkowski
    Nicolaus Copernicus University, Torun, Poland
  • Krzysztof Palczewski
    Pharmacology, Case Western Reserve University, Cleveland, Ohio, United States
  • Grazyna Palczewska
    Polgenix Inc., Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Nathan Alexander, None; Patrycjusz Stremplewski, None; Maciej Wojtkowski, None; Krzysztof Palczewski, 7706863 (P), 8346345 (P), Polgenix, Inc. (C); Grazyna Palczewska, Polgenix Inc. (E)
  • Footnotes
    Support  NIH Grant 1K99EY025007 NIH Grant 1U01EY025451
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 2212. doi:
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      Nathan Alexander, Patrycjusz Stremplewski, Maciej D Wojtkowski, Krzysztof Palczewski, Grazyna Palczewska; Low-power two-photon fluorescence imaging of mouse retinal pigmented epithelium in vivo. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2212.

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

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Purpose : Two-photon fluorescence microscopy (TPM) is an attractive technique for biological imaging. The long wavelength and tissue penetrating benefits of TPM are especially pertinent for non-invasive imaging of the sensitive tissues in the retina. Advances such as lasers with shorter pulses, dispersion correction, and adaptive optics improve the signal during TPM imaging, but the challenge remains to reduce the needed laser power as much as possible to enable safe routine diagnostic imaging in humans, with a target of less than 1 mW. The time and space constraints for two photons to be simultaneously absorbed and induce fluorescence create a challenging problem to obtain an informative signal. Higher laser power increases the amount of photons interacting with present fluorophores but can cause heat damage to tissue.

Methods : We collected images using a range of laser power from 28 mW down to 1.5 mW and investigated the viability of each laser power to provide informative images in conjunction with post-processing image registration and averaging. Images of the retinal pigmented epithelium (RPE) cell layer were collected through the pupil of live, anesthetized two-month old RPE 65-/- knockout mice eyes. Up to 150 images were collected, registered, and averaged for each laser power. All animal procedures and experiments were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University and conformed to recommendations of both the American Veterinary Medical Association Panel on Euthanasia and the Association for Research in Vision and Ophthalmology.

Results : RPE cells can be detected at 1.5 mW of power with a normalized variance of 3075, using the average of the unregistered images as the template image the images are aligned to. When images are registered to an ideal template collected at 31 mW, the normalized variance is 3296, indicating a slightly better resolved image. However, individual images aligned to the ideal template show increased artifacts indicative of over-fitting to the template.

Conclusions : With advances in hardware rapidly leading to improved TPM efficiency in conjunction with adequate image processing, effective imaging below 1.0 mW is foreseeable. In addition, using the unaligned average of collected images as the template produces a more natural registration.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.


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