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ZEISS Scientific Poster Awards
at the Young Researcher Vision Camp 2015

The winners are (1) Niklas Domdei, (2) Kaushikaram Subramanian and (3) Jiri Pergner.

ZEISS recognizes the winners for outstanding scientific contributions and awards excellence in research in vision and ophthalmology

Dr. Thomas Wheeler-Schilling - European Vision Institute EEIG, Niklas Domdei, Kaushikaram Subramanian, Jiri Pergner, Dr. Arne Ohlendorf - ZEISS Vision Science Lab

1st ZEISS AWARDEE – Niklas Domdei

Title:
In vivo imaging and stimulation of human cone photoreceptors with adaptive optics scanning laser ophthalmoscopy

Authors:
Niklas Domdei1, Frank Holz1, Lawrence Sincich2, Wolf Harmening1

Affiliations:
1Department of Ophthalmology, University of Bonn, Germany - 2Department of Vision Sciences, University of Alabama at  Birmingham, AL, USA

2nd ZEISS AWARDEE - Kaushikaram Subramanian

Title:
Tissue level optical benefits of nuclear inversion in mouse photoreceptors.

Authors:
Kaushikaram Subramanian (1), Zuzanna Blaszczak (2), Alfonso Garcia Ulloa (1), Martin Weigert (1), Matthäus Mittasch (1), Irina Solovei (4), Jochen Guck (3), Moritz Kreysing (1).

Affiliations:
(1) The Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. (2) Cavendish Laboratory, Uni versity of Cambridge, Cambridge, UK. (3) Biotechnology Center, Technische Universität Dresden, Dresden, Germany. (4) Department of Biology, LMU München, Germany.

3rd ZEISS AWARDEE – Jiri Pergner

Title:
The Evolution of vertebrate eyes: Insights from Cephalochordates

Authors:
Jiri Pergner, Pavel Vopalensky, Iryna Kozmikova and Zbynek Kozmik

Affiliations:
Department of Transcriptional Regulation, Institute of Molecuar Genetics, Prague, Czech Republic

The Abstracts

In vivo imaging and stimulation of human cone photoreceptors with adaptive optics scanning laser ophthalmoscopy

Niklas Domdei1, Frank Holz1, Lawrence Sincich2, Wolf Harmening1
1Department of Ophthalmology, University of Bonn, Germany - 2Department of Vision Sciences, University of Alabama at Birmingham, AL, USA

Purpose

Adaptive optics scanning laser ophthalmoscopy (AOSLO) can image single photoreceptors in vivo. Combined with real-time eye tracking and correction for chromatic aberrations the AOSLO can be used as a microscopy platform to make single cells optically accessible for functional testing. Due to its scanning nature, visual stimuli can be encoded into the imaging beam with high-speed acousto-optic modulation (AOM), thereby creating an acutely focused visual display directly on the retina. We here characterize the limits and possibilities of such a micro display for the studies of visual function on the level of individual receptor cells in the living eye.

Methods

Measurements were performed with a multi-wavelength AOSLO, with 840 nm light for imaging and 543 nm for stimulation. Since space and time are interlinked in a scanning system, spatial characteristics can be inferred by recording temporal beam intensity modulation. Modulation accuracy for benchmark stimuli (gratings, lines, dots, Gabor patches, complex gray scale images) was measured using a high-speed Si analog photodetector sampled at 1.25 - 5 Gigahertz.  A simple light capture model was used to calculate nominal light delivery under experimental conditions. Stimulus fidelity and visible contrast was validated psychophysically under foveal inspection when higher order aberrations were compensated for.

Results

The smallest full contrast stimuli presentable were on the order of 3 pixels across in raster scanning coordinates. This corresponds to about 2 µm on the retina or ~120ns as the beam continuously sweeps over the retina with a typical raster scanning excursion of 1.2 deg of visual angle. Optical modelling confirms that this size would place almost all light within the dimensions of a single cone inner segment diameter.  Maximum light intensity contrast for extended stimuli achieved in our setup was ~0.99 (Michelson, or about 355:1), a level limited by the extinction ratio of the acousto-optic device used for optical switching. Residual light leak (~4.3 cd/m2 at 543 nm, around 4100 isomerizations per second) through these switches likely saturates any rod photoreceptor contribution, thus AOSLO-based visual psychophysics is currently limited to cone photoreceptor responses.

Conclusions

AOSLO-based micro-stimulation has enough spatial resolution to drive individual cone photoreceptors in the living eye, allowing investigators to probe the relationship between retinal structure and visual function on single cell level. This technique promises to be useful for a host of fundamental and clinical vision research applications.

Acknowledgement

DFG grants HA5323/5-1, HA5323/4-1, HA5323/3-1; Carl Zeiss Förderfonds

Tissue level optical benefits of nuclear inversion in mouse photoreceptors.

Kaushikaram Subramanian (1), Zuzanna Blaszczak (2), Alfonso Garcia Ulloa  (1), Martin Weigert (1), Matthäus Mittasch (1), Irina Solovei (4),  Jochen Guck (3), Moritz Kreysing (1).
(1) The Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. (2) Cavendish Laboratory, University of Cambridge, Cambridge, UK.  (3) Biotechnology Center, Technische Universität Dresden, Dresden, Germany. (4) Department of Biology, LMU München, Germany.

Purpose

It has puzzled biologists for centuries that the vertebrate retina is inverted with respect to its optical function: photons need to traverse multiple layers of living neuronal tissue before detection by photoreceptor cells. Recent findings indicate that cells situated in this light path might circumvent this unfortunate situation by aligning with the light path or minimizing light scattering by adapting their nuclear architecture (Solovei 2009, Kreysing 2010, Blaszczak 2014). My research aims to further explore the effects of the nuclei that account for significant volume fraction of the entire retina (in rodents) and their architecture at a tissue level in enhancing the optical property of retina.

Methods

Using the concept of modulation transfer function we are looking to confirm benefits of nuclear inversion at tissue level. The mechanism of inversion has been recently established (Solovei 2013) where in, the down regulation of proteins associated with nuclear envelope during developmental stages of mice trigger the inversion. In this regard we compare wild type mice vs transgenic mice that express the Lamin B receptor and consequently have conventional nuclear architecture to specifically look at the fingerprint of the spatial frequencies in the transmission spectra of retina due to this inversion. The experiments are complemented by computer simulations of wave optical light propagation through outer nuclear layer as mapped by two-photon excitation microscopy.

Conclusions

We present first experimental and detailed computer simulated evidence for enhanced optical property of the mouse retina at a tissue level owing to its nuclear architecture, stemming from the developmental studies capturing the inversion of nuclear architecture.

The Evolution of vertebrate eyes: Insights from Cephalochordates

Jiri Pergner, Pavel Vopalensky, Iryna Kozmikova and Zbynek Kozmik
Department of Transcriptional Regulation, Institute of Molecuar Genetics, Prague, Czech Republic

Purpose

The evolution of vertebrate camera eye was always puzzling. Even Charles Darwin pointed out in his ground-breaking work On the Origin of Species to topic of eye evolution: „To suppose that the eye, ..., could have been formed by natural selection, seems, I freely confess, absurd in the highest possible degree. Yet reason tells me, that if numerous gradations from a perfect and complex eye to one very imperfect and simple, ..., can be shown to exist; ..., then the difficulty of believing that a perfect and complex eye could be formed by natural selection, though insuperable by our imagination, can hardly be considered real.“Amphioxus (Branchiostoma) and Asymmetron belonging to subphylum Cephalochordates, thanks to their key phylogenetic position, serve as excellent model organisms for studying vertebrate body plan features evolution. In our study we focus on comparison of Cephalochordate and vertebrate eye development. Our previous work, done on Branchiostoma floridae, showed, that highly similar genes are utilized for amphioxus frontal eye (comprising of ~10 photoreceptor and ~10 pigment cells) as well as for vertebrate eye development (comprising of millions of photoreceptor and pigment cells). Currently we are focussing on comparison of frontal eye development between two Cephalochordate genera Branchiostoma and Asymmetron separated in evolution by ~200 million years to get better insight into general rules of Cephalochordate and vertebrate eye development.

Methods

In our study we mainly use whole-mount immunofluorescent and mRNA in situ staining of Branchiostoma and Asymmetron larvae with subsequent confocal microscopy imaging. With this approach we can identife molecular fingerprint of Cephalochordate photoreceptor and pigment cells on a single cell resolution. We strongly benefit from „homemade“ antibodies raised against amphioxus specific antigens.

Conclusions

Our previous and present data show, that development of photoreceptor and pigment cells is highly similar in phylum Chordata from amphioxus to vertebrates. This can help for understanding the evolution of highly complex camera type vertebrate eyes from simple frontal eye of Cephalochordates.

Acknowledgement

We would like to acknowledge GACR, 15-23675S; MSMT, LO1220; MSMT, LO1419 for financial support of this research.

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