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Three‐dimensional quantification of the spatiotemporal co‐evolution of vascular and neuronal networks within intact eyes
Author(s) -
Singh Jasmine,
Seedorf Gregory J.,
Brzezinski Joseph A.,
Shepherd Douglas P.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.857.1
Subject(s) - optical sectioning , computer science , biomedical engineering , artificial intelligence , microscopy , optics , physics , medicine
Retinopathy of prematurity (ROP) is a disease that affects blood vessel development and distribution in the eyes of infants born prematurely. A common method used to investigate structure‐function relationships in ROP is serial sectioning followed by two‐dimensional image analysis. Often, serial sectioning is fraught with inconsistencies due to tissue tearing and folding which may introduce optical artifacts. Of note are the errors that occur when multiple physical sections are computationally reconstructed to quantify the spatial location of fluorescent labels within the original three‐dimensional tissue. In this work we utilize a technique, CLARITY, which renders tissue optically transparent through the establishment of a monomer hydrogel matrix and removal of light scattering lipids to generate optically transparent eyes. To measure fluorescent labels within these intact eyes we have developed a unique digital scanned light sheet microscope (DSLM) specifically designed to quantify fluorescently labeled signaling molecules and structures within CLARITY treated samples. These two techniques combined provide a methodology to quantify the three‐dimensional distribution of key signaling molecules and structures during development of the eye, bypassing issues inherent in serial sectioning, two dimensional imaging and computational reconstruction. Here, we report quantification of the vascular and neuronal network structures in intact control and ROP model rat eyes. Using eyes harvested at multiple time points in development, we provide a comparative analysis evaluating both two dimensional and three dimensional imaging techniques and find that network features in developing eyes are more accurately quantified using our three‐dimensional imaging approach. This paves the way for future studies of the exact timing of changes of signaling and structural development, in three‐dimensions, during the pathogenesis of ROP.