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Alex S. Huang, Phuc Le, Jose M. Gonzalez, Jr., James Tan; In situ Visualization of Extracellular Matrix Biomarkers in Human Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3257.
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In situ 2-photon excitation fluorescence (TPEF) of the human trabecular meshwork (TM) shows beam structures with a heterogeneous autofluorescent signal. This signal is comprised of brightly fluorescent linear fibers on a less intensely fluorescent background. To determine the sources of this heterogeneity we imaged the TM to characterize autofluorescence (AF), collagen second harmonic generation (SHG), and eosin-labeled direct fluorescence of elastin.
Corneoscleral rims retained after corneal transplantation were imaged by TPEF (Leica SP5 and multiphoton laser). Tissue was incubated in 1% eosin for 30 min and then imaged with an excitation wavelength of 850nm. TPEF was collected through multiphoton bandpass filters for AF (525/50nm), collagen SHG (425/20nm), and red fluorescence (635/90nm). Linear regions of relative signal void in SHG images were identified as regions of interest (ROI; Fig. 1B). Quantitative co-localization analysis obtaining Pearson’s (Rr; –1-1) and overlap coefficients (R; 0-1) were performed with the WCIF-ImageJ co-localization plugins to determine if the ROI coincided with eosin fluorescence.
While autofluorescence, SHG, and eosin-fluorescence images of the human TM revealed TM beams and fibers, the images also differed from each other (Fig A-C). SHG images of TM beams where there were regions devoid of signal - (ROI; Fig B/C) - were analyzed. These ROI coincided with eosin staining, representing regions having elastin fibers. Beam regions with SHG signal represented regions with collagen. Quantitative colocalization analyses confirmed this with low Pearson’s and moderate overlap coefficients for ROI and regions of positive eosin signal (Fig B/C).
We present quantitative evidence for the contribution of collagen and elastin to the heterogeneous autofluorescent signal observed in TPEF of TM beams. Interpreting the autofluorescent and SHG signal in this way potentially provides a novel non-invasive method to analyze and identify specific biomarkers in the ex vivo or in vivo TM.
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